Lavendamycin analogs, quinoline-5,8-diones and methods of using them

ABSTRACT

The invention provides novel lavendamycin analogs having the following general formula: 
                 
 
and quinoline-5,8-diones having the following formula: 
                 
 
Methods of making and using and compositions containing these compounds are also disclosed.

This application is a divisional of application Ser. No. 09/515,785,filed Feb. 29, 2000, issued Sep. 3, 2002 as U.S. Pat. No. 6,444,684,which is a divisional of application Ser. No. 08/962,427, filed Oct. 31,1997, issued Feb. 29, 2000 as U.S. Pat. No. 6,030,983, which is acontinuation-in-part of application Ser. No. 08/476,213, filed Jun. 7,1995, issued Jan. 27, 1998 as U.S. Pat. No. 5,712,289, which is acontinuation-in-part of application Ser. No. 08/345,509, filed Nov. 28,1994, issued Jul. 8, 1997 as U.S. Pat. No. 5,646,150, which is acontinuation-in-part of application Ser. No. 08/071,648, filed Jun. 4,1993, issued Jun. 11, 1996 as U.S. Pat. No. 5,525,611, the disclosuresof which are incorporated by reference herein.

This invention was made in part with Government support (NIH grants1-R15-CA54517 and 1-R15-GM37491). The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Lavendamycin (A) was isolated from the fermentation broth ofStreptomyces lavendulae by Doyle and co-workers. See Doyle et al.,Tetrahedron Letters, 22, 4595 (1981) and Gould et al., Fortschr. Chem.Org. Naturst., 41, 77 (1982). Lavendamycin has broad spectrum antitumor,antibacterial and antiviral activity. See, e.g., Shibata et al., J.Antibiot. 33, 1231 (1980); Balitz et al., J. Antibiot. 35, 259 (1982);Boger et al., J. Med. Chem., 30, 1918 (1987).

Lavendamycin methyl ester (B) is also known. It can be prepared byesterification of lavendamycin. Doyle et al., Tetrahedron Letters, 22,4595 (1981). The first total synthesis of lavendamycin methyl ester wasreported by Kende and Ebetino in 1984. See Kende et al., TetrahedronLetters, 25, 923 (1984) and Kende et al., Heterocycles, 21, 91 (1984).They accomplished the synthesis of lavendamycin methyl ester through aBischler-Napieralski condensation of a substituted quinaldic acid with^(b)-methyltryptophan methyl ester followed by cyclization andfunctionalization of the A ring. Boger and his co-workers havesynthesized lavendamycin methyl ester by a Friedlander condensation of afunctionalized aminoaldehyde with a b-carboline followed by othertransformations. Boger et al., J. Org. Chem., 50, 5790 (1985). Formalsyntheses of lavendamycin methyl ester have been reported in Hibino etal., Heterocycles, 23, 261 (1985) and Rao et al., Tetrahedron, 42, 5065(1986). For a recent review of lavendamycin syntheses, see Rao, inRecent Progress In Chemical Synthesis Of Antibiotics, 497-531 (Lukacs etal. eds., 1990).

Hibino's group has reported the synthesis of demethyllavendamycin methylester (C) in Hibino et al., Heterocycles, 20, 1957 (1983). Hibino'sgroup synthesized demethyllavendamycin methyl ester by a Pictet-Spenglertype cyclization of 8-benzyloxyquinoline-2-aldehyde with tryptophanmethyl ester, followed by aromatization and hydrogenation to give an8-hydroxyquinoline intermediate. This intermediate was brominated togive the 5,7-dibromo-8-hydroxyquinoline. Oxidation of the5,7-dibromo-8-hydroxyquinoline yielded the 7-bromoquinolinequinone, andreplacement of the bromine with sodium azide, followed by reduction ofthe azide with sodium hydrosulfite, yielded demethyllavendamycin methylester.

This synthetic scheme based on a Pictet-Spengler type cyclization wasalso used by Hibino's group for the formal synthesis of lavendamycinmethyl ester mentioned above. Hibino et al. indicate that lavendamycinethyl ester can be prepared using this same synthetic scheme. See Hibinoet al., Heterocycles, 23, 261 (1985).

The Pictet-Spengler cyclization approach has further been used byHibino's group to synthesize desaminodesmethyllavendamycin methyl ester(D) and eight other lavendamycin analogs. See Hibino et al., Chem.Pharm. Bull., 34, 1376 (1986). This article reports that the relativemutagenic potency of the lavendamycin analogs was drastically influencedby the nature of the substituent (e.g., methyl and/or bromine) and thatlavendamycin analogs having a methyl group at the 3′ position were moremutagenic.

The structures of lavendamycin and analogs B-D are presented below:

-   -   A: R=H, R′=CH₃, R″=NH₂    -   B: R=CH₃, R′=CH₃, R″=NH₂    -   C: R=CH₃, R′=H, R″=NH₂    -   D: R=CH₃, R′=H, R″=H,

During preliminary work aimed at the total synthesis of lavendamycin,Rao et al synthesized two additional analogs (E) and (F) oflavendamycin. See Rao et al., Indian J. Chem., 23B, 496 (1984). Thestructures of lavendamycin analogs E-F are represented below

-   -   E: R′=CH₃    -   F: R′=H

Lavendamycin is similar structurally to streptonigrin (G). Streptonigrinalso has a broad spectrum of antitumor, antibacterial and antiviralactivity. Balitz et al., J. Antibiot., 35, 259 (1982); Rao et al., J.Am. Chem. Soc., 85, 2532 (1986); Boger et al., J. Med. Chem., 30, 1918(1987). With notable exceptions, lavendamycin has been found to becomparable to, although less potent than, streptonigrin in its observedspectrum of activity. Id.; Balitz et al., J. Antibiot., 35, 259 (1982).The structure of streptonigrin is presented below:

Streptonigrin and several streptonigrin derivatives have beensynthesized. See Driscoll et al., Cancer Chemother. Rep. (Part 2), 4, 1(1974) (four streptonigrin derivatives and 1500 quinones includingseveral quinolinequinone analogs of streptonigrin having varioussubstituents at positions 2, 6 and 7); Rao, Cancer Chemother. Rep. (Part2), 4, 11 (1974) (streptonigrin derivatives and AB and ABC ring analogsthereof; Kende et al., Tetrahedron Lett., 4775 (1978) (tetracyclicaminoquinone possessing full streptonigrin carbon skeleton but withdifferent substituents on the C and D rings); Basha et al., J. Am. Chem.Soc., 102, 3962 (1980) (streptonigrin); Kende et al., J. Am. Chem. Soc.,103, 1271 (1981) (streptonigrin); Weinreb et al., J. Am. Chem. Soc.,104, 53644 (1982) (streptonigrin); Panek et al., Diss. Abs. Int'l, 46,1176B (1985) (streptonigrin); Miyasaka et al., J. Chem. Soc. PerkinTrans., 1, 479 (1986) (streptonigrin 2′-amide derivatives; also mentionsa 7-position amide obtained by high-yield microbial synthesis ofStreptomyces griseus); Tolstikov et al., J. Antibiot., 45, 1020 (1992)(2′-amide, aminodicarboxylic acid and amino sugar derivatives ofstreptonigrin); Tolstikov et al., J. Antibiot., 45, 1002 (1992)(2′-decarboxy-2′-amino streptonigrin); Preobrazhenskaya et al., J.Antibiot., 45, 227 (1992) (streptonigrone from streptonigrin,streptonigrin and streptonigrone 8′-alkyl ethers, and otherstreptonigrin and streptonigrone derivatives); U.S. Pat. No. 3,372,090(ester, 2′-amide, 2′-hydrazide, ether, dihydro, desamino and acetyl(O-acetyl, N-acetyl, tetraacetyl) derivatives of streptonigrin); U.S.Pat. No. 3,804,947 (isopropylidene azastreptonigrin, streptonigrinmonoxime and esters and other derivatives thereof); and JP 61-280490(streptonigrin 2′-amides).

The biological activities of several 2′-position streptonigrinderivatives have been studied. The derivatives include 2′-esters,2′-amides, 2′-hydrazides, and 2-amino acid derivatives. The effects ofthe substituents on the biological activity of streptonigrin varieddepending on the substituent and the type of activity being studied. SeeRivers et al., Cancer Chemotherapy Rep., 46, 17 (1965); Harris et al.,Cancer, 18, 49 (1965); Kremer et al., Biochem. Pharmacol., 15, 1111(1966); Kaung et al., Cancer, 23, 1280 (1969); Inouye et al., J.Antibiot., 38, 1429 (1985); Okada et al., J. Antibiot., 39, 306 (1986);Inouye et al., J. Antibiot., 39, 550 (1986); Okada et al., J. Antibiot.,40, 230 (1987); Take et al., J. Antibiot., 42, 968 (1989); Tolstikov etal., J. Antibiot., 45, 1020 (1992).

The biological properties of streptonigrin, streptonigrin methyl esterand isopropylidene azastreptonigrin have been compared. See Kremer etal., Cancer Chemother. Rep., 51, 19 (1967); Mizuno, Biochem. Pharmacol.,16, 933 (1967); Chaube et al., Cancer Chemother. Rep. (Part 1), 53, 23(1969) and Chirigos et al., Cancer Chemother. Rep. (Part 1), 57, 305(1973). Again, the effects of the substituents varied depending on theactivity being investigated.

The antibacterial activity of streptonigrin, streptonigrin methyl esterand streptonigrin 8′-alkyl ethers has been studied. See Preobrazhenskayaet al., J. Antibiot., 45, 227 (1992). The 8′-alkyl ethers exhibitedslightly greater antibacterial activity than streptonigrin methyl ester,but less than streptonigrin.

A naturally-occurring analog of streptonigrin,10′-desmethoxystreptonigrin, has been discovered. U.S. Pat. No.5,158,960; Liu et al., J. Antibiot., 45, 454-57 (1992). In addition to10′-desmethoxystreptonigrin, U.S. Pat. No. 5,158,960 discloses salts,esters and amides of 10′-desmethoxystreptonigrin. Exemplary esters andamides are those prepared by esterifying the 2′-carboxyl or by formingan amide group at the 2′-position. 10′-Desmethoxystreptonigrin was foundto have anticancer and antimicrobial, particularly broad spectrumantibacterial, activity. U.S. Pat. No. 5,158,960; Liu et al., J.Antibiot., 45, 454-57 (1992). It was also found to be three times moreactive than streptonigrin in an assay for the inhibition of thefarnesylation of ras oncogene p21 protein. Id. U.S. Pat. No. 5,158,960teaches that, since 10′-desmethoxystreptonigrin inhibits thefarnesylation of ras oncogene p21 protein, it may be expected to blockthe neoplastic effect of ras oncogenes in tumor cells.

EP application 185,979 discloses another naturally-occurringstreptonigrin analog which has a hydroxyl group in place of the methoxygroup at the 6-position of streptonigrin. This compound is reported toexhibit only slightly less antitumor activity than streptonigrin, but toexhibit much lower cytotoxicity. This EP application also disclosesderivatives synthesized by making use of the 6-position hydroxyl.

A third naturally-occurring analog of streptonigrin is streptonigrone.Herit et al., J. Antibiot., 38, 516 (1985). Streptonigrone has also beensynthesized from streptonigrin, and streptonigrone 8′-alkyl ethers andother streptonigrone derivatives have been prepared. Preobrazhenskaya etal., J. Antibiot., 45, 227 (1992). Also,2′-decarboxy-2′-aminostreptonigrin, considered to be an analog ofstreptonigrone, has been synthesized. Tolstikov et al., J. Antibiot.,45, 1002 (1992). Streptonigrone and its derivatives have generally beenfound to be inactive or much less active than streptonigrin. See Heritet al., J. Antibiot., 38, 516 (1985); Preobrazhenskaya et al., J.Antibiot., 45, 227 (1992); Tolstikov et al., J. Antibiot., 45, 1002(1992).

Finally, a number of streptonigrin and lavendamycin partial structureshave been synthesized and their biological activities studied in anattempt to determine the minimum potent pharmacophore of streptonigrinand lavendamycin. See Driscoll et al., Cancer Chemother. Rep. (Part 2),4, 1 (1974) (1500 quinones including several quinolinequinone analogs ofstreptonigrin having various substituents at positions 2, 6 and 7; alsofour streptonigrin derivatives); Rao, Cancer Chemother. Rep. (Part 2),4, 11 (1974) (streptonigrin derivatives and AB and ABC ring analogsthereof); Rao, J. Heterocyclic Chem., 12, 725 (1975) (2-phenyl- and2,2-pyridyl-quinoline-5,8-diones); Rao, J. Heterocyclic Chem., 14, 653(1977) (ABC ring portion of streptonigrin and derivatives thereof; Lownet al., Can. J. Chem., 54, 2563 (1976) (2-(o-nitrophenyl)- and2-(o-aminophenyl)-5,8-quinolinediones); Lown et al., Can. J. Biochem.,54, 446 (1976) (substituted 5,8-quinolinequinones related tostreptonigrin); Liao et al., J. Heterocyclic Chem., 13, 1283 (1976) (CDring portion of streptonigrin); Rao et al., J. Heterocyclic Chem., 16,1241 (1979) (ABC ring portion of streptonigrin and analogs); Shaikh etal., Diss. Abs. Inter., 44, 1464B (1983)(2,3-disubstituted-0,1,4-naphthalenediones, 6,7-disubstituted5,8-quinoline, isoquinoline, quinoxoline, quinazoline,phthalazinediones, and2-(o-nitrophenyl)-6,7-disubstituted-5,8-quinolinediones related tostreptonigrin); Boger et al., J. Org. Chem., 50, 5782 (1985) (AB and CDEring portions of lavendamycin); Panek et al., Diss. Abs. Inter., 46,1176B (1985) (streptonigrin and lavendamycin carbon framework); Renaultet al., J. Am. Chem. Soc., 104, 1715 (1985) (5,8-quinazolinediones);Shaikh et al., J. Med. Chem., 29, 1329 (1986) (a series of aza and diazabicyclic quinones related to the AB ring system of streptonigrin); Bogeret al., Heterocycles, 24, 1067 (1986) (streptonigrin and lavendamycin ABring systems); Inouye et al., J. Antibiot., 40, 105 (1987)(6-methoxy-5,8-dihydroquinoline-5,8-dione and6-methoxy-7-methyl-5,8-dihydroquinoline-5,8-dione); Boger et al., J.Med. Chem., 30, 1918 (1987) (various streptonigrin and lavendamycinpartial structures are discussed, including the AB, ABC, CD and CDErings and derivatives thereof); Take et al., J. Antibiot., 40, 679(1987) (quinoline quinones, 7-isoquinoline quinones, indole quinone);Yasuda et al., J. Antibiot, 24, 1253 (1987) (7-amino-2-(2′-pyridyl)quinoline-5,8-quinone-6′-carboxylic acid); Beach, Diss. Abs. Inter, 49,3204-B (streptonigrin isoquinoline analogs); Kitahara et al., Chem.Pharm. Bull., 38, 2841(1990) (8-amino-5,6-quinolinediones); Rao et al.,J. Med. Chem. 34, 1871 (1991) (streptonigrin isoquinoline analogs).

Of particular note is Rao, Cancer Chemother. Rep. (Part 2), 4, 11(1974). This article reports the results of a study of the activity ofseveral streptonigrin derivatives and AB ring analogs which led theauthor to propose a structure (H) for the minimum potent pharmacophoreof streptonigrin.

A tricyclic analog of H (corresponding to the ABC rings ofstreptonigrin) was synthesized and found to be active. Results ofinterest reported in this article are that replacement of the aminefunction at position 7 of streptonigrin by OH or OCH₃ led to loss ofactivity, and streptonigrin derivatives produced by reductiveacetylation or methylation of the amine group at position 7 wereinactive. Esterification of the carboxyl of streptonigrin with a seriesof alcohols gave esters which reportedly showed significant activity.

The fully elaborated streptonigrin CD and lavendamycin CDE ring systems,as well as a number of related synthetic structures, have reportedlyproved inactive in antimicrobial and cytotoxic assays. Boger et al., J.Med. Chem., 30, 1918 (1987). However, none of the AS and ABC ringanalogs of streptonigrin and lavendamycin have been reported to possesscytotoxic, antimicrobial or antitumor activity comparable tostreptonigrin. See id.; Driscoll et al., Cancer Chemother. Rep. Part 2,4, 1(1974). This suggests a role for the CD rings in the activity ofstreptonigrin. See Kende et al., Tetrahedron Lett., 48, 4775 (1978).

The following references also describe the synthesis of streptonigrinand lavendamycin partial structures, but do not discuss the activity ofthe resulting compounds. Liao et al., Angew. Chem. Intern. Edit., 6, 82(1967) (AB ring portion of streptonigrin); Rao et al., J. HeterocyclicChem., 12, 731 (1975) (streptonigrin C ring precursors); Liao et al., J.Heterocyclic Chem., 13, 1063 (1976) (the AB ring portion ofstreptonigrin and the 2-methyl homolog); Hibino et al., J. Org. Chem.,42, 232 (1977) (the AB ring portion of streptonigrin); Wittek et al., J.Org. Chem., 44, 870 (1979) (CD ring portion of streptonigrin); Boger etal., Tetrahedron Lett., 25, 3175 (1984) (the CDE ring portion oflavendamycin); Erickson, Diss. Abs. Inter., 49, 747-B (1988)(4-aminoanthranilic acid, 7-aminoquinaldinic acid,7-amino-5-hydroxyquinaldinic acid); Molina et al., Tetrahedron Lett.,33, 2891 (1992) (1-substituted-^(b)-carbolines). See also, Kaiya et al.,Heterocycles, 27, 64549 (1988) (6- and7-acetylaminoquinoline-5,8-diones); Yanni, Collect. Czech. Chem.Commun., 56, 1919-25 (1991) (6-chloro-7-acylamino-5,8-diones);Klimrovich et al., Khim. Geterotsikl. Soedin., 153941 (1975) (N-acetyland N-propyl derivatives of 7-amino-6-chloro-5,8-quinolinedione); andU.S. Pat. No. 3,933,828.

Reverse transcriptase (RT) of retroviruses are RNA dependent DNApolymerases and play a vital role in the integration of the viral genomeinto host cell DNA and allow for their subsequent replication andpathogenesis. Temin, H., The RNA tumor viruses-background andforeground, Proc. Natl. Acad. Sci. USA, 1972, 69, 1016. Thus, RT is apotential therapeutic target. Nucleoside analog inhibitors of RT, suchas 3′-azido-3′-deoxythymidine (AZT) and dideoxyinosine (ddl), areclinically effective drugs for treating human immunodeficiency virus(HIV) infection. Mitsuya, H.; Broder, S., Toward the rational design ofantiretroviral therapy for HIV infection in “The Human Retroviruses”,Gallo, R.; Jay, G., Eds.; Academic Press Inc.: San Diego, 1991, pp.335-338. Their effectiveness is however, limited by toxicities, whichmay reflect inhibition of cellular polymerases and/or alteration ofnucleoside pools and the emergence of AZT-resistant viral isolates fromAIDS patents. Mitsuya, H.; Yarchoan, R.; Broder, S., Molecular targetsfor AIDS therapy, Science, 1990, 249, 1553. Yarchoan, R.; Broder, S.Anti-retroviral therapy of AIDS and related disorders: generalprinciples and specific development of dideoxynucleosides, Pharmocol.Ther., 1989, 40 329.

Larder, B.; Darby, G.; Richman, D., HIV with reduced sensitivity toZidovudine (AZT) isolated during prolonged therapy, Science, 1989, 243,1731. There is a definite and urgent need to develop selective RTinhibitors that can be used either alone or in combination withnucleoside analogs.

Recent efforts in the search for new drugs that can be used to treat(HIV) disease or AIDS have resulted in the identification of severalfamilies of nonnucleoside inhibitors active against this virus byselective binding to the viral reverse transcriptase. Baba, M.;Declereq, E.; Tanaka, H.; Ubasawa, M.; Takashima, H.; Sekiya, K.; Nitta,I.; Umezu, K.; Nakashima, H.; Mori, S.; Shigeta, S.; Walker, R.;Miyasaka, T., Potent and selective inhibition of human immunodeficiencyvirus type 1 (HIV-1) by 5-ethyl-6-phenylthiouracil derivatives throughtheir interaction with the HIV-1 reverse transcriptase. Proc. Natl.Acad. Sci. USA, 1991, 88, 2356. Goldman, M.; Nunberg, J.; O'Brien, J.;Quintero, J.; Schleif, W.; Freund, K.; Gaul, S.; Saari, S.; Wai, j.;Hoffman, J.; Anderson, P.; Hupe, D.; Emini, E.; Stern, A., Pyridinonederivatives: specific human immunodeficiency virus type I reversetranscriptase inhibitors with antiviral activity. Proc. Natl. Acad. Sci.USA, 1991, 88, 6863. Pauwels, R.; Andries, K.; Desmyter, J.; Schols, D.;Kukla, M.; Breslin, H.; Raeymaekers, A.; VanGelder, J.; Woestenborghs,R.; Heykants, J.; Schellekens, K.; Janssen, M.; DeClereq, E.; Janssen,P., Potent and selective inhibition of HIV-1 replication in vitro by anovel series of TIBO derivatives, Nature (London), 1990, 343,470. Okada,H.; Inouye, Y.; Nakamura, S., Kinetic analysis of inhibition of reversetranscriptase by streptonigrin, J. Antibiotics, 1987, 40, 230. One ofthese families of nonnucleoside inhibitors includes streptonigrin andlavendamycin. Rao, K., Cullen, W., Streptonigrin, an antitumorsubstance 1. Isolation and characterization in “Antibiotics Annual1959-1960”, Welch, H.; Marti-Ibanez, F., Eds.; Medical Encyclopedia,Inc.: New York, 1960, pp. 950-953. Doyle T.; Balitz, D.; Grulich, R.;Nettleton, D.; Gould, S.; Tann, C.; Moens, A., Structure determinationof lavendamycin—A new antitumor antibiotic from Streptomyces lavendulae,Tetrahedron Lett., 1981, 22, 4595. Streptonigrin, an aminoquinolinequinone produced by several Streptomyces species, has a wide spectrum ofantimicrobial, antitumor and anti-viral activities. Inouye, Y.; Okada,H.; Uno, J.; Arai T.; Nakamura, S., Effects of streptonigrin derivativesand sakymicin A on the respiration of isolated rat liver mitochondria,J. of Antibiotics, 1986, 39, 550. This potent antibiotic is a stronginhibitor of the reverse transcriptase of both avian myeloblastosisvirus (AMV) and human immunodeficiency virus reverse transcriptases.Take, Y.; Inouye, S.; Nakamura, S.; Allaudeen, H.; Kubo, A., Comparativestudies of the inhibitory properties of antibiotics on humanimmunodeficiency virus and avian myeloblastosis virus reversetranscriptases and cellular DNA polymerases, J. Antibiotics, 1989, 42,107. Unfortunately, the clinical use of streptonigrin for treating humanmalignancies has been discontinued because of toxicity, primarily bonemarrow depression. Tolstikov, V.; Kozlova, N.; Oreskina, T.; Osipova,T.; Preobrazhenskaya, M.; Sztaricshai, F.; Balzarini, J.; DeClereq, E.,Amides of antibiotic streptonigrin and amino dicarboxylic acids or aminosugars. Synthesis and biologic evaluation, J. Antibiotics, 1992; 45,1020. Hackethal, C.; Golbey, R.; Tan, C.; Karofsky, D.; Burchenal, J.;Clinical observation on the effects of streptonigrin in patients withneoplastic disease, Antibiot. Chemother., 1961 11, 178. More recently,another the biosynthetically related Streptomyces metabolite,lavendamycin described above has been shown in limited studies to becomparable in several of its biological activities to streptonigrin.Balitz, D; Bush, J.; Bradner, W.; Doyle, T.; O'Herron, T.; Nettleton,F., Lavendamycin isolation and antimicrobial and antitumor testing, J.Antibiotic, 1982, 35, 259. Unfortunately, lavendamycin itself alsoappears to be toxic and will probably not be clinically useful either.Boger, D.; Yasuda, M.; Mitscher, L.; Drake, S.; Kitos, P.; Thompson, S.,Streptonigrin and lavendamycin partial structures. Probes for theminimum, potent pharmacophore of stretonigrin, lavendamycin andsynthetic quinoline-5,8-diones, J. Med. Chem., 1987, 30, 1918.

SUMMARY OF THE INVENTION

The invention provides a lavendamycin analog having the followingformula (I):

wherein,

Y is H, OR¹¹, SR¹¹, N(R¹¹)₂, NR¹¹N(R¹¹)₂, a halogen atom, NO₂, CN,

an alkyl, aryl, cycloalkyl, alkynyl, alkenyl or heterocyclic residue,each of which may be substituted or unsubstituted,

-   -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, which may be the same or        different, each is independently H, a halogen atom, NO₂, CN,        OR¹³, SR¹³, N(R¹³)₂,        an alkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl,        heterocyclic, heteroalkenyl or heteroalkynyl residue, each of        which may be substituted or unsubstituted,    -   R⁹ is H,        an alkyl, cycloalkyl, aryl, alkenyl, alkynyl or heterocyclic        residue, each of which may be substituted or unsubstituted,    -   R¹⁰, R¹¹ and R¹³, which may be the same or different, each is        independently H or an alkyl, cycloalkyl, alkenyl, alkynyl, aryl        or heterocyclic residue, each of which may be substituted or        unsubstituted,    -   R¹² is H, N(R¹¹)₂, OR¹¹, SR¹¹, NR¹¹N(R¹¹)₂, OR¹⁴ N(R¹¹)₂, or an        alkyl, cycloalkyl, aryl, alkenyl, alkynyl or heterocyclic        residue, each of which may be substituted or unsubstituted, and    -   R¹⁴ is an alkylene residue, and        salts of these lavendamycin analogs.

In one aspect of the invention, R¹ is halogen atom, preferably Cl.

In another aspect of the invention, the compound has the followingformula:

-   -   wherein R1 is a halogen atom, and    -   R⁴ is H, a halogen atom, NO₂, CN, an alkyl, aryl, cycloalkyl,        alkenyl, alkynyl, heteroalkyl, heterocyclic, heteroalkenyl or        heteroalkynyl residue, each of which may be substituted or        unsubstituted. Preferably, R¹ is Cl and R⁴ is CH₃.

The invention also provides a method of preparing these lavendamycinanalogs which comprises reacting an aldehyde having the followingformula (K):

with a tryptophan analog of the formula (L):

wherein X, Y and R¹ through R⁹ are as defined above.

The lavendamycin analogs of the invention have antitumor andantimicrobial (antibacterial, antiviral and antiparasitic) activity. Inparticular, certain of the lavendamycin analogs of the invention haveunexpected selective activity against ras^(K) tumor cells.

The invention, therefore, provides methods of treating animals having atumor or suffering from a microbial infection which comprisesadministering to the animals an effective amount of a lavendamycinanalog of the invention or a pharmaceutically-acceptable salt thereof.The invention also provides the use of the above-described lavendamycincompounds in treating cancer. The invention also provides pharmaceuticalcompositions comprising a lavendamycin analog, or apharmaceutically-acceptable salt thereof, in combination with apharmaceutically acceptable carrier.

The lavendamycin analogs of the invention also have anti-HIV ReverseTranscriptase (HIV-RT) activity by themselves and preferably incombination with 3′ azido-3′-deoxythymidine (AZT). Accordingly, theinvention provides the use of the lavendamycin analogs in treating HIVinfection and a method and composition for treating HIV infection withthe lavendamycin analogs and with combinations of the lavendamycinanalogs in combination with AZT.

The invention also provides a method of inhibiting the growth ofmicrobes comprising contacting the microbe with a lavendamycin analog ofthe invention, or a salt thereof. For instance, the lavendamycin analogsof the invention may be added to liquids to inhibit microbial growth inthem. The lavendamycin analogs may also be formulated into disinfectantpreparations useful for inhibiting microbial growth on surfaces.

The invention further provides quinoline-5,8-diones having the followingformula (V):

wherein, X, R¹, R² and R³ are as defined above and Z is CH₃ or CHO.

In one aspect of the invention R¹ is a halogen atom, preferably Cl.

In another aspect of the invention, the compound has the followingformula:

-   -   wherein R¹ is a halogen atom, preferably Cl, and Z is either CHO        or CH₃.

The inventions also provides a method of preparing these quinolinedioneswhich comprises reacting a 1-silyloxy-azadiene having the followingformula (N):

with a bromoquinone of the formula (O):

wherein X, R¹, R², R³ are as defined above.

The quinolinediones of the invention have antitumor activity. They arealso useful for the synthesis of the lavendamycin analogs of theinvention.

The invention, therefore, provides methods of treating animals having atumor which comprises administering to the animals an effective amountof a quinolinedione of the invention or a pharmaceutically-acceptablesalt thereof. The invention also provides pharmaceutical compositionscomprising a quinoline dione, or a pharmaceutically-acceptable saltthereof, in combination with a pharmaceutically-acceptable carrier.

The invention also provides methods of treating cancer usinglavendamycin methyl ester and its analogs, preferably6-chlorolavendamycin methyl ester.

Finally, the invention provides methods of treating an animal sufferingfrom HIV infection with the lavendamycin analogs andquinoline-5,8-diones described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: Graphs of percent survival versus drug concentration.

FIG. 2: Graph of percent increase in footpad size versus time.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The lavendamycin analogs of the invention have general formula I givenabove. The quinolinediones of the invention have general formula V givenabove. In the definition of substituents X, Y and R¹ through R⁹ offormulas I and V, the following terms have the following meanings.

“Alkyl” refers to straight or branched chain alkyl residue containingfrom 1 to 20 carbon atoms. Alkyl residues include methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,2-methylbutyl, 2,2-dimethylpropyl, isoamyl, n-hexyl, 2-methylpentyl,2,2-dimethylbutyl, n-heptyl, 2-methylhexyl, n-octyl, etc.

“Aryl” refers to a residue comprising at least one aromatic ring of 5 or6 carbon atoms. Aryl residues include phenyl, tolyl, biphenyl, naphthyl,etc.

“Cycloalkyl” refers to an aliphatic ring having from 3 to 8 carbonatoms. Cycloalkyl residues include cyclopropyl, cyclopentyl, cyclohexyl,etc.

“Alkenyl” refers to a straight or branched chain alkyl residue whichcontains from 1 to 20 carbon atoms and at least one carbon—carbon doublebond. Alkenyl residues include vinyl, allyl, 1,1-dimethyl allyl, etc.

“Alkynyl” refers to a straight or branched chain alkyl residue whichcontains from 1 to 20 carbon atoms and at least one carbon—carbon triplebond. Alkynyl residues include ethynyl, propenyl, etc.

“Alkylene” refers to a straight or branched chain alkylene residuecontaining from 1 to 20 carbon atoms. Alkylene radicals includemethylene, ethylene, propylene, etc.

“Heteroalkyl” refers to an alkyl containing one or more heteroatomsselected from oxygen, sulfur and nitrogen.

“Heterocyclic” refers to a cycloalkyl or aryl containing one or moreheteroatoms selected from oxygen, sulfur and nitrogen. Heterocyclicresidues include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isothiazolyl,isoxazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, etc.

“Heteroalkenyl” refers to an alkenyl containing one or more heteroatomsselected from oxygen, sulfur and nitrogen.

“Heteroalkynyl” refers to an alkynyl containing one or more heteroatomsselected from oxygen, sulfur and nitrogen.

The alkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl,heterocyclic, heteroalkenyl and heteroalkynyl residues may beunsubstituted or substituted with one or more substituents. Suitablesubstituents include R^(X), NH₂, R^(X)NH, (R^(X))₂N, CN, N₃. NO₂, OH,halogen (Cl, Br, F. I), SH, R^(X)S, R^(X)SO₂, R^(X)SO, R^(X)O, COOH,COOR^(X), COR^(X), CHO, and CON(R^(X))₂, wherein R^(X) is an alkyl,cycloalkyl, aryl, alkenyl, alkynyl or heterocyclic residue. Preferredsubstituents are halogen atoms.

Preferred lavendamycin analogs of the invention are those having theformula (J):

wherein X, Y, R⁴ and R⁶ are defined above.

More preferred are lavendamycin analogs of formula J wherein:

-   -   R⁴ is H or an alkyl, and    -   R⁶ is H, a halogen atom or OR¹³,        wherein R¹⁰, R¹² and R¹³ are defined above. R¹⁰ is preferably an        alkyl or substituted alkyl, Y is preferably        and when Y is so defined, R¹² is preferably N(R¹¹)₂, OR¹¹ or        OR¹⁴N(R¹¹)₂. When R¹² is so defined, R¹¹ is preferably H or an        alkyl or cycloalkyl. When R¹¹ is an alkyl, it preferably        contains from 2 to 20 carbon atoms, most preferably from 4 to 20        carbon atoms.

Preferred quinolinediones are those of formula V wherein

-   -   R¹⁰ is an alkyl or substituted alkyl,    -   R¹=H,    -   R²=H, and    -   R³=H.

Certain lavendamycin analogs and quinoline-diones of the presentinvention may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptableacids. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedby reacting a purified compound of the invention in its free base formwith a suitable organic or inorganic acid and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthalate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.Other salts of the lavendamycin analogs and quinolinediones containing abasic functional group may be used for non-therapeutic uses.

In other cases, the compounds of the invention may contain one or moreacidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively nontoxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can be preparedby reacting the purified compound in its free acid form with a suitablebase, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. Other salts ofthe lavendamycin analogs and quinolinediones containing an acidicfunctional group may be used for non-therapeutic uses.

The lavendamycin analogs I of the invention may be synthesized by anefficient procedure, the final step of which is a Pictet-Spenglercondensation of an aldehyde of the following formula K:

with a tryptophan analog of the formula (L):

-   -   wherein X, Y and R′ through R⁹ are as defined above. See        Equation 1 below:

Typical reactions involve the combination of equimolar amounts of K andL in a solvent. Suitable solvents include aromatic hydrocarbons such asxylene, toluene, mesitylene, and anisole. The reactants are heated untilthe reaction is complete. Generally, the temperature will be about 60°C. or higher, and the reaction will take about 1 hour or more to reachcompletion. The lavendamycin analog is obtained in the form of a solidby: (1) cooling the reaction mixture; (2) concentrating the solution andthen cooling; or (3) concentration and treatment with other solvents inwhich the lavendamycin analogs are not soluble. Such solvents includeethyl acetate, pentane, hexane, cyclohexane, petroleum ether, diethylether, and acetone.

Aldehyde K can be prepared by the selenium dioxide oxidation of thecorresponding 2-methylquinoline-5,8-dione (M) in refluxing dioxane-H₂O.

The 2-methylquinoline-5,8-diones M can be prepared by the Diels-Aldercondensation of a 1-silyloxy-azadiene (N) with a bromoquinone (O) inrefluxing chlorobenzene for 24 hours. See Equation 2 below. Bromoquinone(O) can be prepared according to the method described in Kelly et al.,J. Org. Chem., 48, 3849 (1983) (see Example 27).

The azadiene N can be prepared by the reaction of a silyloxyamine (P)with a ketone (Q) in dichloromethane at room temperature for 48 hours inthe presence of molecular sieves. See Equation 3 below.

Some of the 2-methylquinoline-5,8-diones M can also be prepared by theoxidation of the corresponding acylamido compounds (R) with potassiumdichromate in glacial acetic acid. See Equation 4 below.

The acylamido compounds R may be prepared by the reduction of the5,7-dinitro-8-hydroxy-2-methylquinolines (S) by molecular hydrogen inthe presence of palladium on carbon followed by treatment with thedesired anhydride (T). See Equation 5.

Tryptophan and its analogs suitable for use in the method of theinvention are available commercially or can be made by methods known inthe art. For instance, tryptophan analogs 35, 38, 42 and 43 (see TableVI) are commercially available in their salt forms. Also, the followingtryptophan analogs useful in the practice of the invention arecommercially available from Sigma Chemical Co., St. Louis, Mo.:NA-BOC-L-tryptophan p-nitrophenyl ester; N-CBZ-L-tryptophanp-nitrophenyl ester; DL-tryptophanamide hydrochloride; L-tryptophanbenzyl ester hydrochloride; DL-tryptophan butyl ester hydrochloride;DL-tryptophan ethyl ester hydrochloride; L-tryptophan ethyl ester;D-tryptophan methyl ester hydrochloride; L-tryptophan methyl ester;DL-tryptophan octyl ester hydrochloride; 5-methoxy-DL-tryptophan;5-fluorotryptamine hydrochloride; 6-fluorotryptamine;DL-4-fluorotryptophan; 5-fluoro-DL-tryptophan; 6-fluoro-DL-tryptophan;5-hydroxy-D-tryptophan; 5-hydroxy-DL-tryptophan; 5-hydroxy-L-tryptophan;5-hydroxy-DL-tryptophan ethyl ester hydrochloride. Other tryptophananalogs are available from other sources or can be made by methods knownin the art.

For instance, tryptophan esters can be prepared by reacting tryptophanor an appropriately functionalized tryptophan with an alcohol to givethe desired ester. See Equation 6

Alkylaminoalkyl esters can be prepared by protecting the amine on thetryptophan (or functionalized tryptophan analog) and then reacting theprotected tryptophan with the appropriate alkylaminoalkyl halide asdescribed in Matao Kanoaka et al., J. Phar. Soc. of Japan, 95, 231(1975). See Equation 7 below.

Tryptophan esters of tertiary alcohols can be prepared by the method ofRosowsky et al., J. Med. Chem., 24, 1450 (1981) as shown in Equation 8:

wherein R^(X) is an alkyl, and each R^(X) may be the same or different.

Tryptophan aryl esters can be prepared by the methods of Castro et al.Synthesis, 413 (1977) and Rosowsky et at. J. Med. Chem., 24, 1450 (1981)as shown in Equation 9:

Finally, tryptophan esters and amides can be prepared by theneutralization of the commercially available salts with, e.g., ammoniumhydroxide, followed by extractions

In addition to the method of preparing the lavendamycin analogs of theinvention just described, the lavendamycin analogs can also be preparedby means of a Bischer-Napieralski condensation of an appropriatelyfunctionalized quinoline-2-carboxylic acid (U) with an appropriatelyfunctionalized tryptophan L. This method is similar to the methoddescribed in Kende et al, Tetrahedron Letters, 25, 923 (1984).

The lavendamycin analogs of the invention are useful as antitumor,antibacterial, antiviral and antiparasitic agents. For instances, theycan be used to treat bacterial infections caused by both gram-positiveand gram-negative bacteria, for example, bacteria of the genusStaphylococcus (such as Staphylococcus aureus), Streptococcus (such asStreptococcus agalactine and Streptococcus faecalis), Micrococcus (suchas Micrococcus luteus), Bacillus (such as Bacillus subtilis), Listerella(such as Listerella monocytogenes), Escherichia (such as Escherichiacoli), Klebsiella (such as Klebsiella pneumoniae), Proteus (such asProteus mirabilis and Proteus vulgains), Salmonella (such as Salmonellatyphosa), Shigella (such as Shigella sonnei), Enterobacter (such asEnterobacter aerogenes), Serratia (such as Serratia marcescens),Pseudomonas (such as Pseudomonas aeruginosa), Acinetobacter such asAcinetobacter anitratus), Nocardia (such as Nocardia autotrophica), andMycobacterium (such as Mycobacterium fortuitum).

The lavendamycin analogs of the invention also have antiviral activity.For instance, they can be used to treat viral infections caused by theRetroviridae (e.g., HIV-1, HIV-2, HTLV-I and HTLV-II), Herpesviridae(e., herpes simplex, varicella zoster, Epstein-Barr virus, and herpesgenitalia), Hepadnaviridae (e.g., hepatitis B), Picornaviridae h.,hepatitis A virus and poliomyelitis virus), hepatitis non A non 8 virus,Orthomyxoviridae (eq., influenza virus), Poxyiridae (e.g., variola virusand vaccinia virus), Flaviviridae (e.g., yellow fever virus),Rubiviridae (ea., rubella virus), Paramyxoviridae (eq., measles,parainfluenza, mumps and canine distemper viruses), Rhabdoviridae (eq.,rabies virus), Papovaviridae, and Adenoviridae.

Moreover, some of the lavendamycin analogs, namely compounds B describedabove, and compounds 1, 2, 12, 16, 17, 20 and 21 described below, havebeen shown to have anti-HIV reverse transcriptase activity (See Example57 below). These compounds were shown to have relatively low toxicity.These compounds were also shown to have remarkable additiveeffectiveness when combined with 3′-azido-3′-deoxythymidine.

The lavendamycin analogs can also be used to treat parasitic infections,including those caused by Amoeba, Giardia, Babesia, Balantidium,Eimeriorina, Entamoeba, Histomonas, Naegleria, Nosema, Plasmodium,Toxoplasma, Trypanosoma and Trypanosomatidae.

The lavendamycin analogs also have antitumor activity. They may be usedto treat a variety of tumors, including ovarian, colon, breast,cervical, esophageal, glioblastoma, neuroblastoma, stomach, kidney,skin, lung, pancreatic, seminoma, melanoma, bladder, thyroid, myeloidand lymphoid tumors. They have been found to be especially effectiveagainst solid tumors and malignant tumors.

In particular, certain of the lavendamycin analogs of the invention haveunexpectedly been found to be selectively active against ras^(K) tumorcells. By “selectively active,” it is meant that these lavendamycinanalogs are more cytotoxic towards the ras^(K) tumor cells than towardsnormal cells. Example 47 describes a method of determining whichlavendamycin analogs are selectively active against ras^(K) tumor cells.Other methods are known in the art.

The lavendamycin analogs of the invention which have been found to beselectively active against ras^(K) tumor cells are those compounds offormula J wherein:

-   -   R¹⁰ is an alkyl,    -   R¹² is OR¹¹ or OR⁴N(R¹¹)₂,    -   R¹¹ is an alkyl,    -   R⁴ is H or an alkyl, and    -   R⁶ is H.        Three of these compounds have been found to exhibit        unprecedented highly selective activity against ras^(K) tumor        cells. They are compounds of formula J wherein:    -   R¹⁰ is CH₃,    -   R¹² is OR¹¹,    -   R¹¹ is methyl, isoamyl, or n-octyl,    -   R⁴ is CH₃ when R¹¹ is methyl, and R⁴ is H when R¹¹ is isoamyl or        n-octyl, and    -   R⁶ is H.

The ras^(K) oncogene has been associated with the causation of a largenumber of human solid tumors, including 90% of pancreatic cancers, 60%of colon cancers and 30% of breast cancers. Prior to the presentinvention there were no effective drugs available to treat patients withsolid tumors whose malignant phenotype was maintained by the ras^(K)oncogene.

One lavendamycin analog, namely 6-chlorolavendamycin methyl ester hasalso shown activity in the NCI Hollow Fiber Assay For Preliminary InVivo Testing (see Example 56 below).

The quinolinediones also have antitumor activity. They may be used totreat a variety of tumors, including ovarian, colon, breast, kidney,lung, prostate, central nervous system, melanoma, and lymphoid tumors.They have been found to be especially effective against solid tumors andmalignant tumors.

Treatment of bacterial, viral and parasitic infections according to theinvention includes both mitigation, as well as elimination, of theinfection. Treatment of tumors includes maintaining or reducing the sizeof, or eliminating, the tumor.

To treat an animal suffering from a microbial (bacterial, viral orparasitic) infection, an effective amount of a lavendamycin analog ofthe invention, or a pharmaceutically-acceptable salt thereof, isadministered to the animal. Animals treatable according to the inventioninclude mammals such as dogs, cats, other domestic animals, and humans.

To treat an animal suffering from an HIV infection, an effective amountof a lavendamycin analog, or a pharmaceutically-acceptable salt thereof,is administered to the animal, preferably in combination with othercompounds such as nucleoside analog inhibitors of HIV reversetranscriptase, most preferably in combination with3′-azido′3′-deoxythymidine (AZT).

To treat an animal having a tumor, an effective amount of a lavendamycinanalog or quinoline dione of the invention, orpharmaceutically-acceptable salts thereof, is administered to theanimal. Animals treatable according to the invention include mammalssuch as dogs, cats, other domestic animals, and humans.

Effective dosage forms, modes of administration and dosage amounts ofthe lavendamycin analogs and quinolinediones may be determinedempirically, and making such determinations is within the skill of theart. It is understood by those skilled in the art that the dosage amountwill vary with the activity of the particular lavendamycin analog orquinolinedione employed, the severity of the microbial infection ortumor, the route of administration, the rate of excretion of thelavendamycin analog or quinolinedione, the duration of the treatment,the identity of any other drugs being administered to the animal, theage, size and species of the animal, and like factors well known in themedical and veterinary arts. In general, a suitable daily dose of acompound of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect.Exemplary daily dosages of the lavendamycin analogs are those within therange of from about 1 mg/kg/day to about 3 g/kg/day, preferably from 10mg/kg/day to 500 mg/kg/day. Exemplary daily dosages of the quinolinediones are those within the range of from about 1 mg/kg/day to about 3g/kg/day, preferably from 10 to 500 mg/kg/day. However, the total dailydosage of the lavendamycin analog or quinolinedione will be determinedby an attending physician or veterinarian within the scope of soundmedical judgment. If desired, the effective daily dose of a lavendamycinanalog or a quinoline-dione, or pharmaceutically-acceptable saltsthereof, may be administered as two, three, four, five, six or moresub-doses, administered separately at appropriate intervals throughoutthe day.

The compounds of the present invention may be administered to an animalpatient for therapy by any suitable route of administration, includingorally, nasally, rectally, intravaginally, parenterally,intracisternally and topically, as by powders, ointments or drops,including buccally and sublingually. The preferred routes ofadministration are orally and parenterally.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise one or more of the compounds ofthe invention as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient.

Pharmaceutical formulations of the present invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. Regardless of theroute of administration selected, the compounds of the presentinvention, are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art.

The amount of active ingredient (a lavendamycin analog, quinolinedione,or pharmaceutically-acceptable salts thereof which will be combined witha carrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration andall of the other factors described above. The amount of activeingredient which will be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing into association a compound of the presentinvention with the carrier and, optionally, one or more accessoryingredients are known in the art. In general, the formulations areprepared by uniformly and intimately bringing into association acompound of the present invention with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia), each containing a predeterminedamount of a compound of the present invention as an active ingredient. Acompound of the present invention may also be administered as bolus,electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (a lavendamycin analog, a quinolinedione, orpharmaceutically-acceptable salts thereof) is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar—agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract and/or in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient (a lavendamycin analog, a quinolinedione, orpharmaceutically-acceptable salts thereof), the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar—agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating acompound of the invention in a proper medium, such as an elastomericmatrix material. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate-controlling membrane or dispersingthe compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The pharmaceutical compositions of the present invention may also beused in the form of veterinary formulations, including those adapted forthe following: (1) oral administration, for example, drenches (aqueousor non-aqueous solutions or suspensions), tablets, boluses, powders,granules or pellets for admixture with feed stuffs, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular or intravenous injection as, for example,a sterile solution or suspension or, when appropriate, by intramammaryinjection where a suspension or solution is introduced into the udder ofthe animal via its teat; (3) topical application, for example, as acream, ointment or spray applied to the skin; or (4) intravaginally, forexample, as a pessary, cream or foam.

The lavendamycin analogs of the invention may also be employed asantimicrobial (antibacterial, antiviral, antiparasitic) agents useful ininhibiting the growth of microbes. “Inhibition of growth” as used hereinincludes killing of the microbes. For instance, the compounds of theinvention may be used to inhibit the growth of microbes present on asurface or in a medium outside a living host. The invention, therefore,provides a method for inhibiting the growth of a bacterium, virus orparasite comprising the step of contacting the microbe with alavendamycin analog of the invention, or a salt thereof, in an amounteffective for the inhibition. Thus, the lavendamycin analogs of theinvention may be employed, for example, as disinfectants for surfacetreatments or as preservatives for a variety of solid and liquid mediasusceptible to microbial growth. Effective amounts of a lavendamycinanalog for such uses may be determined empirically by methods known tothe skilled artisan.

EXAMPLES Examples 1-46 Synthesis of Lavendamycin Analogs

Examples 1-46 describe the synthesis of 22 lavendamycin analogs. Thesynthesis of 8 quinoline-diones is also described. The following generalprocedures were utilized in Examples 1-46. Melting points (uncorrected)were determined on a Thomas Hoover capillary apparatus and are indegrees Celsius. Infrared (IR) spectra were obtained with a Nicolet 5ZDXspectrophotometer. Proton magnetic resonance (¹H NMR) spectra wereobtained with a Varian Gemini-200 spectrophotometer in CDCl₃ or DMSO-d₆with tetramethylsilane (TMS) as internal standard. Mass spectra (MS)were obtained with a Hewlett-Packard 5980A instrument fitted with avacummetrics solid probe or with a Kratos MS 50. Elemental analyses wereperformed by Midwest Microlabs, Ltd.

Examples 1-22 Synthesis of 7-N-Acyllavendamycins

The 22 lavendamycin analogs which were prepared have formula J:

wherein:

-   -   R¹⁰ is an alkyl or substituted alkyl,    -   R⁴ is H or an alkyl,    -   R⁶ is H, a halogen atom or OR¹³,    -   R¹² is N(R¹¹)₂, OR¹¹ or OR¹⁴ N(R¹¹)₂,    -   R¹¹ is H, an alkyl or a cycloalkyl,    -   R¹³ is an alkyl, and    -   R¹⁴ is an alkylene.        Table I contains a list of the lavendamycin analogs and        specifies the substituents for each analog.

The lavendamycin analogs were prepared by reacting an appropriatelyfunctionalized aldehyde K with an appropriately functionalizedtryptophan analog L as described above (see Equation 1 above). Thereaction conditions and yields for the 22 lavendamycin analogs arelisted in Table I, and the procedures used to make 11 of the analogs aredescribed in detail below. Table II gives the ¹H NMR, MS and highresolution MS (HRMS) values for the 22 analogs.

7-N-Acetyllavendamycin Methyl Ester (Compound 1. Table I): A stirredmixture of aldehyde 23 (prepared as described in Example 23) (12.02 mg,0.05 mmol) and tryptophan ester 39 (prepared as described in Behforouzet al., J. Heterocycl. Chem., 25, 1627 (1988); see Example 39 below)(11.6 mg, 0.05 mmol) and dry xylene (16 mL) under argon was slowlyheated (3 hours) to boiling and then refluxed for 19 hours. The solutionwas concentrated to 5 mL and then cooled. The pure orange crystals ofthe 7-N-acetyllavendamycin methyl ester were filtered. A yield of 17.8mg, 78.4%, was obtained. The melting point (mp) was >300° C. IR (KBr)3473 (br), 3310, 3121, 1728, 1708, 1680, 1645, 1588, 1504, 1398, 1335,1307, 1229, 1124, 738 cm⁻¹; ¹H NMR (CDCl₃)·2.38 (s, 3, COCH₃), 3.19 (s,3, ArCH₃), 4.07 (s, 3, CO₂CH₃), 7.39 (t, 1, J=8 Hz., H-11′), 7.65 (t, 1,J=8 Hz., H-10′), 7.74 (d, 1, J=8 Hz., H-9′), 8.00 (s, 1, H-6), 8.35 (d,1, J=8 Hz., H-12′), 8.41 (brs, 1, CONH), 8.5 (d, 1, J=7 Hz., H-3), 9.1(d, 1, J=7.5 Hz., H-4); 11.85 (br s, 1, NH); MS m/e (relative intensity)454 (M+, 68), 396 (14), 394 (25), 352 (17), 106 (36), 91 (100), 77 (14),54 (14); HRMS, m/e for C₂₅H₁₈N₄O₆ calculated 454.1277, found 454.1277.

7-N-Acetyldemethyllavendamycin (Compound 2, Table I): L-tryptophan (20.4mg, 0.1 mmol) and anisole were stirred and heated in a 60-70° C. oilbath. Then, a mixture of 7-N-acetamido-2-formylquinoline-5,8-dione (23)(prepared as described in Example 23) (24.4 mg, 0.1 mmol) and 20 mlanisole was added dropwise over a one-hour period. The mixture washeated in the oil bath at 120° C. for 23.5 hr. and rotoevaporated todryness. The residue was chromatographed on a silicon gel column (4 gsilica gel, column 13 cm×10 cm), and eluted with CH₂Cl₂ (50 ml),methanol: CH₂Cl₂ (2.5:100, 100 ml) to give orange crystals (0.6 mg.,yield 1.4%).

7-N-Chloroacetyllavendamycin Methyl Ester (Compound 7, Table I): In a 40ml. round-bottomed flask equipped with a reflux condenser, argon-filledballoon and a magnetic bar,7-chloroacetamido-2-formylquinoline-5,8-dione (24) (prepared asdescribed in Example 24) (0.0278 g., 0.10 mmol), b-methyltryptophanmethyl ester (39) (prepared as described in Behforouz et al., J.Heterocycl. Chem., 25, 1627 (1988); see Example 39 below) (0.0232 g.,0.10 mmol), and 40 ml. of dried, distilled xylene were slowly heated to135° C. over a three-hour period. The mixture was heated at thistemperature for an additional 16 hours. A light brown precipitate beganto form at the maximum temperature. The completion of the reaction wasmonitored by TLC. The mixture was filtered hot to remove theprecipitated impurities, and the filtrate was allowed to cool, resultingin the formation of an orange-red solid. This solid was filtered,collected, and dried under vacuum. The yield of the product was 0.0226g. (46%): mp >300° C.; ¹H NMR (DMSO-d₆)* 3.08 (3H, s, ArCH₃), 3.97 (3H,s, COCH₃), 4.64 (2H, s, C-7NHCOCH₂Cl), 7.42 (1H, dd, J=8.4 Hz., J=8.4Hz., C-11′H), 7.69 (1H, dd, J=8.4 Hz., J=8.4 Hz., C-10′H), 7.71 (1H, d,J=8.4 Hz., C-9′H), 7.75 (1H, s, C-6H), 8.40 (1H, d, J=8.4 Hz., C-12′H),8.50 (1H, d, J=8.4 Hz., C-3H), 8.64 (1H, d, J=8.4 Hz., C-4H), 10.58 (1H,br s, C-7NH), 11.94 (1H, br s, NH); IR (KBr) v_(max) 3342, 3106, 3010,2950, 1679, 1652, 1589, 1507, 1492, 1339, 1307, 742 cm⁻¹; electronimpact MS (EIMS), m/e (relative intensity) 488/490 (M⁺, 2.25/1, 16), 454(96), 412 (base), 394 (36), 380 (20), 352 (88), 335 (29); HRMS, m/e forC₂₅H₁₇ClN₄O₅ calculated 488.088748, found 488.087100.

7-N-Chloroacetyldemethyllavendamycin Isoamyl Ester (Compound 9, TableI): In a 100 ml. three-necked, round-bottomed flask equipped with areflux condenser, flowing argon and a magnetic bar,7-chloro-acetamido-2-formylquinoline-5,8-dione (24) (prepared asdescribed in Example 24) (0.0500 g., 0.18 mmol), L-tryptophan isoamylester (37) (prepared as described in Example 37) (0.0490 g., 0.18 mmol),and 75 ml. of dried and distilled xylene were slowly heated to 76EC overa five-hour period. Upon the first detection of the formation of alight-colored precipitate, the mixture was filtered hot. The filtratewas concentrated under reduced pressure to about 10 ml. The filtrate wasstored in a refrigerator overnight during which a considerable amount ofa dark orange-brown solid formed. This was filtered and washed with coldethyl acetate giving 25.5 mg. of the crude product (a yield of 27%). Thefiltrate was further concentrated which, upon cooling, yielded anadditional 15 mg. of the product (16% yield, total yield 43%): mp.280-284EC; ¹H NMR (CDCl₃)*1.05 (6H, d, J=6.3 Hz., (CH₃)₂), 1.76-1.95(3H, m, COOCH₂CH₂CH), 4.29 (2H, s, C-7NHCOCH₂Cl), 4.52 (2H, t, J=6.8Hz., COOCH₂), 7.39 (1H, dd, J=8.0 Hz., J=8.0 Hz., C-11′H), 7.66 (1H, dd,J=8.0 Hz., J=8.0 Hz., C-10′H), 7.78 (1H, d, J=8.0 Hz., C-9′H), 7.95 (1H,s, C-6H), 8.23 (1H, d, J=8.0 Hz., C-12′H), 8.54 (1H, d, J=8.3 Hz.,C-3H), 8.93 (1H, s; C-3′H), 9.18 (1H, d, J=8.3 Hz., C-4H), 9.56 (1H,brs, C-7NH), 11.76 (1H, brs, NH); IR (KBr) v_(max) 3336, 2957, 2929,2908, 2871, 1713, 1680, 1651, 1588, 1520, 1337, 1308, 1265, 1219, 1119,738 cm⁻¹; EIMS, m/e (relative intensity) 530/532 (M⁺, 2.4/1, 45), 496(58), 480 (6), 454 (39), 416 (83), 382 (95), 366 (15), 340 (base); HRMS,m/e for C₂₈H₂₃ClN₄O₅ calculated 530.135698, found 530.135781.

7-N-Chloroacetyldemethyllavendamycin Octyl Ester (Compound 10. Table I):In a 50 ml. three-necked round-bottomed flask equipped with a refluxcondenser, flowing argon and a magnetic bar,7-chloroacetamido-2-formylquinoline-5,8-dione (24) (prepared asdescribed in Example 24) (0.0279 g., 0.10 mmol), L-tryptophan octylester (38) (prepared as described in Example 38) (0.0316 g., 0.10 mmol),and 32 ml. of dried and distilled xylene were slowly heated to 100° C.over a six-hour period. This temperature was maintained for anadditional 2.5 hours. During the last three hours, a brown precipitateformed. TLC of the mixture indicated the major spot to be the desiredproduct. The mixture was filtered hot to remove the precipitatedimpurities, and the filtrate was rotoevaporated to dryness affording0.0301 g. of a dark-red solid which ¹H NMR indicated was crude product(yield of 53%). The crude product was further purified byrecrystallization with ethyl acetate: mp 215-217° C.; ¹H NMR(CDCl₃)*0.88 (3H, t, J=6.8 Hz., (CH₂)₇CH₃)), 1.23-1.70 (10′H,(CH₂)₅CH₃)), 1.84-1.95 (2H, m, COOCH₂CH₂), 4.29 (2H, s, C-7NHCOCH₂Cl),4.48 (2H, t, J=6.8 Hz., COOCH₂), 7.39 (1H, dd, J=7.6 Hz., J=7.6 Hz.,C-11′H), 7.66 (1H, dd, J=7.6 Hz., J=7.6 Hz., C-10′H), 7.79 (1H, d, J=7.6Hz., C-9′H), 7.97 (1H, s, C-6H), 8.24 (1H, d, J=7.6 Hz., C-12′H), 8.56(1H, d, J=8.4 Hz., C-3H), 8.95 (1H, s, C-3′H), 9.21 (1H, d, J=8.4 Hz.,C-4H), 9.58 (1H, br s, C-7NH), 11.79 (1H, br s, NH); IR (KBr) v_(max)3331, 2955, 2926, 2854, 1708, 1680, 1652, 1588, 1519, 1337, 1307, 1264,1219, 738 cm⁻¹; EIMS, m/e (relative intensity) 573 (M+, 1), 538 (10),496 (35), 382 (53), 366 (30), 340 (base); HRMS, m/e for C₃₅H₄₀ClN₄O₇S₂calculated 727.202696, found 727.200500.

7-N-Butyryllavendamycin Methyl Ester (Compound 11, Table I): In a 25 ml.round-bottomed flask equipped with a reflux condenser, argon-filledballoon, and a magnetic bar, 7-butyramido-2-formylquinoline-5,8-dione(26) (prepared as described in Example 26) (0.0272 g., 0.10 mmol),b-methyltryptophan methyl ester (39) (prepared as described in Example39) (0.0232 g., 0.10 mmol), and 30 ml. of dried and distilled xylenewere slowly heated to 130° C. over a three-hour period. This temperaturewas maintained for an additional 16 hours, during which a slight amountof a light brown precipitate formed. The completion of the reaction wasmonitored by TLC. The mixture was filtered hot to remove theprecipitate. The filtrate was then concentrated under reduced pressureto the point of crystallization and then allowed to cool. The orange-redsolid was filtered and then dried under vacuum. The weight of the solidwas 0.0210 g. (a yield of 44%): mp. 270-273° C.; ¹H NMR (CDCl₃)*1.06(3H, t, J=7.6 Hz., C-7NHCOCH₂CH₂CH₃), 1.75-1.90 (2H, m,C-7NHCOCH₂CH₂CH₃), 2.52 (2H, t, J=7.6 Hz., C-7NHCOCH₂CH₂CH₃), 3.16 (3H,s, Ar—CH₃), 4.07 (3H, s, COCH₃), 7.36 (1H, dd, J=8.0 Hz., J=8.0 Hz.,C-11′H), 7.63 (1H, dd, J=8.0 Hz., J=8.0 Hz., C-10′), 7.70 (1H, d, J=8.0Hz., C-9′H), 7.92 (1H, s, C-6H), 8.31 (1H, d, J=8.0 Hz., C-12′H), 8.34(1H, br s, C-7NH), 8.41 (1H, d, J=8.3 Hz. C-3H), 9.01 (1H, d, J=8.3 Hz.,C-4H), 11.76 (1H, br s, NH); IR (KBr) v_(max) 3316, 3199, 2959, 2933,2874, 1724, 1710, 1679, 1646, 1586, 1501, 1335, 1307, 1227 cm¹; EIMS,m/e (relative intensity) 482 (M+, base), 466 (11), 450 (13), 422 (32),380 (9), 352 (35), HRMS, m/e for C₂₇H₂₂N₄O₅ calculated 482.159020, found482.160385.

7-N-Butyryldemethyllavendamycin Isoamyl Ester (Compound 13, Table l): Ina 50 ml. three-necked, round-bottomed flask equipped with a refluxcondenser, flowing argon, and a magnetic bar,7-butyramido-2-formylquinoline-5,8-dione (26) (prepared as described inExample 26) (0.0279 g., 0.10 mmol), L-tryptophan isoamyl ester (37)(prepared as described in Example 37) (0.0274 g., 0.10 mmol), and 30 ml.of dried and distilled xylene were slowly heated to reflux over afour-hour period. This temperature was maintained for an additional 17hours. A small amount of an orange-brown solid was formed. Thecompletion of the reaction was monitored by TLC. The mixture wasfiltered hot to remove the precipitate which was not the product. Thefiltrate was concentrated under reduced pressure to approximately 2 ml.Upon cooling, this solution yielded a bright orange precipitate whichweighed 0.0166 g. (32% yield). The mother liquor was evaporated todryness to yield an additional amount of crude product that weighed0.0198 g. (38% yield), giving a total yield of 70%: mp. 234-235E C; ¹HNMR (CDCl₃)*1.04 (6H, d, J=6.8 Hz., (CH₃)₂), 1.06 (3H, t, J=7.4 Hz.,C-7NHCOCH₂CH₂CH₃), 1.75-1.95 (5H, m, C-7NHCOCH₂CH₂, COOCH₂CH₂CH), 2.53(2H, t, J=7.4 Hz., C-7NHCOCH₂), 4.52 (2H, t, J=6.8 Hz., COOCH₂), 7.39(1H, dd, J=7.8 Hz., J=7.8 Hz., C— 11′H), 7.66 (1H, dd, J=7.8 Hz., J=7.8Hz., C-10′H), 7.74 (1H, d, J=7.8 Hz., C-9′H), 7.99 (1H, s, C-6H), 8.24(1H, d, J=7.8 Hz., C-12′H), 8.41 (1H, br s, C-7NH), 8.55 (1H, d, J=8.4Hz., C-3′H), 8.94 (1H, s, C-3′H), 9.18 (1H, d, J=8.4 Hz., C4H), 11.80(1H, br s, NH); IR (KBr) v_(max) 3369, 3346, 3313, 2960, 2935, 2873,1728, 1715, 1700, 1681, 1646, 1587, 1494, 1335, 1307, 1261, 1221, 1119,739 cm⁻¹; EIMS, m/e (relative intensity) 524 (69), 454 (6), 410 (base),394 (5), 340 (22); HRMS, m/e for C₃₀H₂₈N₄O₅ calculated 524.205970, found524.207494.

7-N-Butyryldemethyllavendamycin Octyl Ester (Compound 14, Table I): In a50 ml. three-necked, round-bottomed flask equipped with a refluxcondenser, flowing argon, and a magnetic bar,7-N-butyramido-2-formylquinoline-5,8-dione (26) (prepared as describedin Example 26) (0.0230 g., 0.085 mmol), L-tryptophan octyl ester (38)(prepared as described in Example 38) (0.0270 g., 0.085 mmol), and 30ml. of dried and distilled xylene were slowly heated to 85EC over a fivehour period. This temperature was maintained for an additional 16 hours.During this period a small amount of a dark precipitate appeared. Thecompletion of the reaction was monitored by TLC. The mixture wasfiltered hot to remove the precipitated impurities, and the filtrate wasrotoevaporated to dryness affording 0.0386 g. of a dark-red solid which¹H NMR indicated was crude product (yield of 80%). The crude product wasfurther purified by recrystallization from ethyl acetate: mp. 166-171EC; ¹H NMR (CDCl₃)*0.86 (3H, t, J=6.8 Hz., (CH₂)₇CH₃), 1.06 (3H, t, J=7.3Hz., C-7NHCOCH₂CH₂CH₃), 1.20-1.60 (10′h, (CH₂)₅CH₃), 1.65-1.95 (4H, mC-7NHCOCH₂CH₂, COOCH₂C2H), 2.53 (2H, t, J=7.3 Hz., C-7NHCOCH₂), 4.47(2H, t, J=6.8 Hz., COOCH₂), 7.36 (1H, dd, J=8.0 Hz., J=8.0 Hz., C-11′H),7.63 (1H, dd, J=8.0 Hz., J=8.0 Hz., C-10′H), 7.65 (1H, d, J=8.0 Hz.,C-9′H), 7.93 (1H, s, C-6H), 8.19 (1H, d, J=8.0 Hz., C-12′H), 8.35 (1H,brs, C-7NH), 8.44 (1H, d, J=8.4 Hz., C-3H), 8.87 (1H, s, C-3′H), 9.08(1H, d, J=8.4 Hz., C-4H), 11.67 (1H, br s, NH); IR (KBr) v_(max) 3317,2957, 2926, 2855, 1728, 1704, 1679, 1646, 1587, 1504, 1333, 1307, 1260,1222, 1118, 739 cm⁻¹; EIMS, m/e (relative intensity) 566 (M+, 14), 550(45), 534 (15), 496 (6), 410 (12), 394 (base), 378 (22), 340 (17); HRMS,m/e for C₃₃H₃₄N₄Os calculated 566.252921, found 566.251991.

7-N-Acetyldemethyllavendamycin Carboxamide (Compound 16. Table l):7-N-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as described inExample 23) (42.3 mg 0.15 mmol), L-tryptophan amide (43) (prepared asdescribed in Example 43) (30.44 mg, 0.015 mmol) and anisole (60 ml) wereheated slowly in an oil bath from room temperature to 160° C. over a2.5-hr. period, and then held 160° C. for 14 hr. The reaction mixturewas cooled in the refrigerator for 3 hr. Then it was filtered to give alemon yellow solid. The solid was washed with hexane and dried to give39.6 mg., a yield of 62%.

7-N-Acetyldemethyllavendamycin Methyl Ester (Compound 17. Table l):7-N-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as described inExample 23) (44.8 mg, 0.2 mmol), L-tryptophan methyl ester (35)(prepared as described in Example 35) (43.6 mg, 0.2 mmol), dry xylene(90 ml) were mixed. The mixture was stirred and slowly heated in an oilbath from room temperature to 140° C. over a 3-hr period and from 140°C. to 154° C. over a 20-hr period. The reaction mixture was allowed tocool to room temperature to give 53 mg (yield of 60%) of a yellow solid.

7-N-Acetyllavendamycin Isoamyl Ester (Compound 18, Table l):7-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as described inExample 23) (44.8 mg, 0.2 mmol), b-methyltryptophan isoamyl ester (36)(prepared as described in Example 36) (57.6 mg, 0.2 mmol) and 64 ml ofdry xylene were stirred and heated slowly in an oil bath from roomtemperature to 140EC over a 3-hour period and was then kept at 140° C.for 1 hour. The hot reaction mixture was filtered to give an orangesolid. The filtrate afforded more product upon cooling. The total weightobtained of the product was 60.2 mg, a 59% yield.

7-N-Acetyllavendamycin N-N-Dimethylaminoethyl Ester (Compound 19. Tablel): 7-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as describedin Example 23) (317 mg, 0.13 mmol) was dissolved in 16 ml of dry anisoleand heated to 80° C. b-methyltryptophan N,N-dimethylaminoethyl ester(41) (prepared as described in Example 41) (37.5 mg, 0.13 mmol) wasadded with stirring, and the stirred mixture was heated at 100° C. for5.5 hr. The reaction mixture was allowed to cool to room temperature andthe solid was filtered. The filtrate was distilled under vacuum todryness. Ethyl acetate (3 ml) was added, and the mixture was stirred.Upon filtration 24.5 mg (a yield of 36%) of an orange solid wasobtained.

7-N-Acetyldemethyllavendamycin Isoamyl Ester (Compound 20. Table l):7-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as described inExample 23) (244 mg, 0.1 mmol), L-tryptophan isoamyl ester (37)(prepared as described in Example 37) (239 mg, 1 mmol) and xylene (64ml) were mixed. The mixture was stirred and slowly heated in an oil bathfrom room temperature to 110° C. over a one-hour period, and then from110° C. to 140° C. over a 3-hr period. The hot reaction mixture wasfiltered to remove the brown impurity. The filtrate was evaporated undervacuum. The residue was treated with ethyl-acetate/hexane to give anorange solid (213 mg, 42% yield).

7-N-Acetyldemethyllavendamycin n-Octyl Ester (Compound 21. Table l):7-Acetamido-2-formylquinoline-5,8-dione (23) (prepared as described inExamples 23-26) (44.8 mg, 0.2 mmol), L-tryptophan N-octyl ester (38)(prepared as described in Example 38) (63.2 mg, 0.2 mmol) and 64 ml ofdry xylene were stirred and slowly heated in an oil bath from roomtemperature to 150° C. over a 3-hr period and then maintained at 150° C.for 2 h. The hot mixture was filtered, and the brown solid wascollected. More brown solid was obtained from the filtrate upontreatment with ethylacetate and hexane (total yield 51 mg, 48%).

7-N-Acetyldemethyllavendamycin N,N-Dimethylaminoethyl Ester (Compound22. Table l): 7-Acetamido-2-formylquinoline-5,8-dione (23) (prepared asdescribed in Example 23) (12.2 mg, 0.05 mmol), L-tryptophanN,N-dimethylaminoethyl ester (40) (prepared as described in Example 40)(14 mg, 0.05 mmol) and 18 ml of anisole were heated at 100° C. for 27 h.The mixture was allowed to cool to room temperature and was filtered toremove impurities. The filtrate was evaporated under reduced pressure,and the resulting residue was dissolved in 6 ml of CHCl₃ and purified bythick layer chromatography on alumina. A pure orange solid (5 mg, 20%yield) was obtained.

TABLE I Compd. R¹⁰ Y R⁴ R⁶ Solvent Hours (° C.) Yield (%) 1 CH₃ CO₂CH₃CH₃ H Xylene 19 (reflux) 78.4 2 CH₃ CO₂H H H Anisole 23.5 (120N) 1.4 3CH₃ CH₂CH₂ H H Xylene 3 (25N-reflux) 59 CO₂CH CH₂ 1 (reflux) CH₃CH₂ 4CH₃ CO₂(CH₂)₃CH₃ H OCH₃ Anisole 6 (70-90N) 66 5 CH₃ CO₂(CH₂)₃CH₃ H FAnisole 5 (120N) 49 6 CH₃ H H H Anisole 3 (25-160N) 54 16 (160N) 7 ClCH₂CO₂CH₃ CH₃ H Xylene 16 (135N) 46 8 ClCH₂ CO₂(CH₂)₃CH₃ H H Xylene 3(<76N) 20 5.75 (76N) 9 ClCH₂ CO₂(CH₂)₂CH(CH₃)₂ H H Xylene 5 (#76N) 43(crude) 10 ClCH₂ CO₂(CH₂)₇CH₃ H H Xylene 6 (<100N) 53 (crude) 2.5 (100N)11 CH₂(CH₂)₃ CO₂CH₃ CH₃ H Xylene 15 (130N) 44 12 CH₂(CH₂)₂ CO₂(CH₂)₃CH₃H H Xylene 55 (130N) 30 13 CH₂(CH₂)₂ CO₂(CH₂)₂CH(CH₃)₂ H H Xylene 4(25N-reflux) 70 (crude) 17 (reflux) 14 CH₂(CH₂)₂ CO₂(CH₂)₇CH₃ H H Xylene5 (<85N) 80 (crude) 16 (85N) 15 (CH₃)₂CH CO₂(CH₂)₂CH₃ H H Xylene 8(125N) 65.2 16 CH₃ CONH₂ H H Anisole 2.5 (<160N) 62 14 (160N) 17 CH₃CO₂CH₃ H H Xylene 3 (<140N) 60 20 (154N) 18 CH₃ CO₂CH₂CH₂CH(CH₃)₂ CH₃ HXylene 3 (<140N) 59 1 (140N) 19 CH₃ CO₂(CH₂)₂N(CH₃)₂ CH₃ H Anisole 5.5(100N) 36 1 (<140N) 20 CH₃ CO₂CH₂CH₂CH(CH₃)₂ H H Xylene 1 (140N) 42 3(<150N) 21 CH₃ CO₂(CH₂)₂CH₃ H H Xylene 3 (<180N) 48 2 (150N) 22 CH₃CO₂CH₂CH₂N(CH₃)₂ H H Anisole 27 (100N) 20

TABLE II Compound R¹⁰ Y R⁴ R⁶ ¹H, NMR MS HRMS 1 CH₃ CO₂CH₃ CH₃ H 2.38(s,3H), 3.22(s, 3H), 4.10(s, 3H), 454 (M⁺, 68), 396 C₂₅H₁₆N₄O₅ 7.42(dd,J=7.6Hz, 1H), 7.68(dd, (14), 394 (25), Calc. 454.1277 J=7.6Hz, 1H),7.77(d, J=8.2Hz, 352 (17), 106 Found 454.1277 1H), 8.00(s, 1H), 8.38(d,J=8.2Hz, (36), 91 (100), 77 1H), 8.44(s, 1H), 8.53(d, J=8.2Hz, (14), 54(14) 1H), 9.13(d, J=8.2Hz, 1H), 11.88 (br s, 1H) 2 CH₃ CO₂H H H 2.37(s,3H), 7.65(dd, 1H), 7.70(dd, 1H), 8.0(s, 1H), 8.13(d, 1H), 8.21 (d, 1H),8.45(br s, 1H), 8.57(d, 1H), 8.62(s, 1H), 9.11(d, 1H), 11.65(br s, 1H) 3CH₃ CH₂—CH₂ H H 1.66-2.17(m, 10H), 2.37(s, 3H), 5.20 508 (M⁺, 35), 426C₂₉H₂₄N₄O₅ CO₂CH CH₂ (m, 1H), 7.41(dd, 1H), 7.68(dd, 1H), (12), 382(base), Calc. 508.174670 CH₂—CH₂ 7.75(d, 1H), 7.98(s, 1H), 8.27(d, 366(15), 340 Found 508.173290 1H), 8.42(br s, 1H), 8.56(d, 1H), (25), 312(5), 8.95(s, 1H), 9.22(d, 1H), 11.8(br s, 283 (4), 256 (6), 1H). 154(4), 130 (4) 4 CH₃ CO₂(CH₂)₃CH₃ H OCH₃ 1.04(t, 3H), 1.6-1.85(m, 4H),2.38(s, 512/513 (M⁺, C₂₈H₂₄N₄O₆ 3H), 4.0(s, 3H), 4.35(t, 2H), 7.33(d,base), 412 (30), Calc. 512.169585 1H), 7.66(d, 1H), 7.67(s, 1H), 7.99(s,397 (6), 355 (8) Found 512.168362 1H), 8.42(br s, 1H), 8.57(d, 1H),8.93(s, 1H), 9.22(d, 1H), 11.7(br s, 1H) 5 CH₃ CO₂(CH₂)₃CH₃ H F 1.07(s,3H), 1.89-1.25(m, 4H), 2.37 (s, 3H), 4.51(t, 2H), 7.19(s, 1H), 7.92(d,1H), 7.94(d, 1H), 8.02(s, 1H), 8.45(br s, 1H), 8.62(d, 1H), 8.93(s, 1H),9.25(d, 1H), 11.88(br s, 1H) 6 CH₃ H H H 2.37(s, 3H), 7.35(dd, 1H),7.68(dd, 382/383 (M⁺ C₂₂H₁₂N₄O₃ 1H), 7.71(d, 1H), 7.99(s, 1H), 8.13base), 340 (58), Calc. 340.096026 (d, 1H), 8.21(d, 1H), 8.45(br s, 1H),313 (24), 284 Found 340.095347 8.61(d, 1H), 8.56(d, 1H), 9.10(d, (12),244 (19), 1H), 11.64(br s, 1H) 140 (10) 7 ClCH₂ CO₂CH₃ CH₃ H (DMSO)3.08(s, 3H), 3.97(s, 3H), 488/490 (M⁺ C₂₅H₁₇ClN₄O₅ 4.64(s, 2H), 7.42(dd,J=8.4Hz, 2.25/1, 16), 454 Calc 488.088748 8.4Hz, 1H), 7.69(dd, J=8.4,8.4Hz, (95), 412 (base), Found 488.087100 1H), 7.71(d, J=8.4Hz, 1H),7.75(s, 394 (36), 380 1H), 8.40(d, J=8.4Hz, 1H), 8.50(d, (20), 352 (88),J=8.4Hz, 1H), 8.64(d, J=8.4Hz, 1H), 335 (29) 10.58(br s, 1H), 11.94(brs, 1H) 8 ClCH₂ CO₂(CH₂)₃CH₃ H H 1.06(t, 3H), 1.85-1.95(m, 4H), 4.25 482(M⁺−Cl+1, (s, 2H), 4.50(t, 2H), 7.35-7.45(m, 41.2), 466 (base), 1H),7.65-7.73(t, 1H), 7.80-7.84 440 (46.6), 424 (t, 1H), 8.01(s, 1H),8.25-8.79(d, (32.4), 382 1H), 8.58-8.62(d, J=8.34Hz, 1H), (30.2), 3648.98(s, 1H), 9.23-9.27(d, J= (15.3), 352 8.38Hz, 1H), 9.62(br s, 1H),11.8 (23.0), 350 (7.9), (br s, 1H) 340 (300), 326 (25.9), 325 (15.0),281 (41.7), 207 (94.1), 182 (14.3), 181 (14.2), 168 (59.9), 129 (14.9),117 (47.5), 115 (21.3) 9 ClCH₂ CO₂(CH₂)₂CH(CH₃)₂ H H 1.05(d, J=6.3Hz,6H), 1.76-1.95(m, 530/532 (M⁺ C₂₈H₂₃ClN₄O₅ 3H), 4.29(s, 2H), 4.52(t,J=6.8Hz, 2.4/1, 45) 496 Calc 530.135698 2H), 7.39(dd, J=8.0, 8.0Hz, 1H),(58), 480 (6), Found 530.135781 7.66(dd, J=8.0, 8.0Hz, 1H), 7.78(d, 454(39), 416 J=8.0Hz, 1H), 7.95(s, 1H), 8.23(d, (83), 382 (95), J=8.0Hz,1H), 8.54(d, J=8.3Hz, 366 (15), 340 1H), 8.93(s, 1H), 9.18(d, J=8.3Hz,(base). 1H), 9.56(br s, 1H), 11.76(br s, 1H). 10 ClCH₂ CO(CH₂)₇CH₃ H H0.88(t, J=6.8Hz, 3H), 1.23-1.70(m, 573 (M⁺, 1), 538 M+C₄H₁₀O₂S₂ 10H),1.84-1.95(m, 2H), 4.29(s, 2H), (10), 496 (35), C₃₅H₄₀ClN₄O₅S₂ 4.48(t,J=6.8Hz, 2H), 7.39(dd, J= 382 (53), 366 Calc 727.202696 7.6, 7.6Hz, 1H),7.66(dd, J=7.6Hz, (30), 340 (base) Found 727.200500 7.6Hz, 1H), 7.79(d,J=7.6Hz, 1H), 7.97(s, 1H), 8.24(d, J=7.6Hz, 1H), 8.56(d, J=8.4Hz, 1H),8.95(s, 1H), 9.21(d, J=8.4Hz, 1H), 9.58(br s, 1H), 11.79(br s, 1H) 11CH₃(CH₂)₂ CO₂CH₃ CH₃ H 1.06(t, J=7.6Hz, 3H), 1.75-1.90(m, 482 (M⁺, base)C₂₇H₂₂N₄O₅ 2H), 2.52(t, J=7.6Hz, 2H), 3.16(s, 466 (11), 450 Calc482.159020 3H), 4.07(s, 3H), 7.36(dd, J=8.0, (13) 422 (32), Found482.160385 8.0Hz, 1H), 7.63(dd, J=8.0, 8.0Hz, 380 (9), 352 1H), 7.70(d,J=8.0Hz, 1H), 7.92(s, (35) 1H), 8.31(d, J=8.0Hz, 1H), 8.34(br s, 1H) 12CH₃(CH₂)₂ CO₂(CH₂)₃CH₃ H H 1.10(t, 6H), 1.6(m, 2H), 1.85-2.00 510 (M⁺,69), 494 C₂₉H₂₆N₄O₅ (m, 4H), 2.55(t, 2H), 4.50(t, 2H), (21.4), 446 Calc510.090320 7.38(t, 1H), 7.65-7.70(m, 2H), 7.97 (11.8), 417 Found510.091661 (s, 1H), 8.20-8.24(d, 1H), 8.39 (11.5), 410 (br s, 1H),8.50-8.54(d, J=8.3Hz, (base), 394 1H), 8.92(s, 1H), 9.13-9.23(d, (35.9),379 J=8.2Hz, 1H), 11.75(br s, 1H) (10.3), 340 (38.4) 339 (16.7), 217(130) 13 CH₃(CH₂)₂ CO₂(CH₂)₂CH(CH₃)₂ H H 1.04(d, J=6.8Hz, 6H), 1.06(t,J= 524 (69), 454 (6), C₃₀H₂₆N₄O₅ 7.4Hz, 3H), 1.75-1.95(m, 5H), 2.53 410(base), 394 Calc 524.205970 (t, J=7.4Hz, 2H), 4.52(t, J=6.8Hz, (5), 340(22) Found 524.207494 2H), 7.39(dd, J=7.8, 7.8Hz, 1H), 7.66(dd, J=7.8,7.8Hz, 1H), 7.74(d, J=7.8Hz, 1H), 7.99(s, 1H), 8.24(d, J=7.8Hz, 1H),8.41(br s, 1H), 8.55(d, J=8.4Hz, 1H), 8.94(s, 1H), 9.18(d, J=8.4Hz, 1H),11.80(br s, 1H) 14 CH₃(CH₂)₂ CO₂(CH₂)₇CH₃ H H 0.86(t, J=6.8Hz, 3H),1.06(t, J= 566 (M⁺, 14), 550 C₃₃H₃₄N₄O₅ 7.3Hz, 3H), 1.20-1.60(m, 10H),(45), 534 (15), Calc. 566.252921 1.65-1.95(m, 4H), 2.53(t, J=7.3Hz, 496(6), 410 Found 566.251991 2H), 4.47(t, J=6.8Hz, 2H), 7.36(dd, (12), 394(base), J=8.0, 8.0Hz, 1H), 7.63(dd, J=8.0, 378 (22), 340 8.0Hz, 1H),7.65(d, J=8.0Hz, 1H), (17) 7.93(s, 1H), 8.19(d, J=8.0Hz, 1H), 8.35(br s,1H), 8.44(d, J=8.4Hz, 1H), 8.87(s, 1H), 9.08(d, J=8.4Hz, 1H), 11.67(brs, 1H) 15 (CH₃)₂CH CO₂(CH₂)₃CH₃ H H 1.04(t, J=7.3Hz, 7.32Hz, 3H),1.33(d, 510/512 (M⁺, C₂₉H₂₈N₄O₅ J=6.68Hz, 6H), 2.73(m, J=6.76Hz, base),494 (37.1), Calc. 510.190320 6.88Hz, 1H), 4.49(t, J=6.88, 6.58Hz, 492(22.4), 469 Found 510.188750 2H), 7.39(d, J=7.3Hz, 1H), 7.41(dd, (5.8),440 (6), J=7.3Hz, 1H), 7.73(dd, J=7.3Hz, 410 (18.1), 394 1H), 7.99(s,1H), 8.24(d, J=8Hz, (35.4), 337 (9.7) 1H), 8.49(s, 1H), 8.55(d, J=8.4Hz,1H), 8.94(s, 1H), 9.18(d, J=8.4Hz, 1H), 11.8(br s, 1H) 16 CH₃ CONH₂ H H2.23(s, 3H), 7.39(t, 1H), 7.70(t, 1H), 425 (M⁺, base), C₂₃H₁₅N₅O₄7.79(d, 1H), 8.46(br s, 1H), 8.57(d, 409 (50), 382 Calc. 425.112404 1H),9.05(s, 1H), 9.06(d, 1H), 9.61(s, (23), 366 (31), Found 425.11150 1H),9.77(br s, 2H), 10.15(d, 1H), 340 (25) 12.39(br s, 1H) 17 CH₃ CO₂CH₃ H H2.38(s, 3H), 4.12(s, 3H), 7.40(dd, 443 (M⁺3, base), C₂₄H₁₆O₅N₄ 1H),7.76(dd, 1H), 7.76(d, 1H), 441 (23), 440 Calc. 440.112070 8.03(s, 1H),8.27(d, 1H), 8.48(br s, (81), 439 (14), Found 440.111984 1H), 8.62(d,1H), 9.03(s, 1H), 9.26 438 (39) (d, 1H), 11.82(br s, 1H) 18 CH₃CO₂CH₂CH₂CH(CH₃)₂ CH₃ H 1.06(d, 6H), 1.8-1.9(m, 3H), 2.37(s, 510 (M⁺68),396 C₂₉H₂₆O₅N₄ 3H), 3.19(s, 3H), 4.53(t, 2H), 7.40 (90), 354 (21), Calc.510.150320 (dd, 1H), 7.60(dd, 1H), 7.70(d, 1H), 281 (16) Found510.187804 7.97(s, 1H), 8.36(d, 1H), 8.39(br s, 1H), 8.50(d, 1H),9.10(d, 1H), 11.80 (br s, 1H) 19 CH₃ CO₂CH₂CH₂N(CH₃)₂ CH₃ H 2.37(s, 3H),2.43(s, 6H), 2.8(t, 2H), 514 (M+3⁺ 20), 3.2(s, 3H), 4.6(t, 2H), 7.40(dd,1H), 425 (17), 397 7.69(dd, 1H), 7.74(d, 1H), 7.97(s, (21), 387 (16),1H), 8.3(d, 1H), 8.4(br s, 1H), 8.5(d, 355 (30), 207 1H), 9.10(d, 1H),11.80(br s, 1H) (17), 155 (51), 147 (48), 119 (base) 20 CH₃CO₂CH₂CH₂CH(CH₃)₂ H H 1.06(d, 6H), 1.8-1.9(m, 3H), 2.38(s, 496 (M⁺55),382 C₂₈H₂₄O₅N₄ 3H), 4.54(t, 2H), 7.41(dd, 1H), 7.69 (100), 340 (17)Calc. 496.174610 (dd, 1H), 7.71(d, 1H), 8.01(s, 1H), Found 496.1755708.27(d, 1H), 8.45(br s, 1H), 8.58(d, 1H), 8.97(s, 1H), 9.22(d, 1H),11.80 (br s, 1H) 21 CH₃ CO₂(CH₂)₇CH₃ H H 0.89(t, 3H), 1.33(m, 10H),1.95(m, 538 (M⁺, 55), 382 C₃₁H₃₀N₄O₅ 2H), 2.37(s, 3H), 4.5(t, 2H),7.43(dd, (100), 366 (5), Calc. 538.221620 1H), 7.70(dd, 1H), 7.73(d,1H), 8.0 340 (16) Found 538.222007 (s, 1H), 8.26(d, 1H), 8.44(br s, 1H),8.6(d, 1H), 8.97(s, 1H), 9.2(d, 1H), 11.8(br s, 1H) 22 CH₃CO₂CH₂CH₂N(CH₃)₂ H H 2.38(s, 3H), 2.47(s, 6H), 2.93(t, 2H), 391 (12),279 4.63(t, 2H), 7.44(dd, 1H), 7.69(dd, (10), 167 (25), 1H), 7.70(d,1H), 7.98(s, 1H), 8.24 150 (17), 149 (d, 1H), 8.42(br s, 1H), 8.5(d,1H), (base), 113 8.96(s, 1H), 9.17(d, 1H), 11.79(br s, (26) 1H)

Examples 23-26 Preparation of Quinolinedione Aldehydes

The starting materials for preparing the lavendamycin analogs ofExamples 1-22 include the 7-N-acyl-2-formylquinoline-5,8-diones offormula K:

-   -   wherein

R¹⁰ is an alkyl or substituted alkyl,

-   -   R¹=H,    -   R²=H, and    -   R³=H.        These aldehydes were prepared by the selenium dioxide oxidation        of the corresponding 7-N-acyl-2-methylquinoline-5,8-diones in        refluxing dioxane-H₂O as described above. Table III lists four        such aldehydes prepared by this method and the reaction times,        yields, ¹H NMR, MS and elemental analysis for these aldehydes.        The procedures used to synthesize three of these aldehydes are        described in detail below.

7-Acetamido-2-Formylquinoline-5.8-Dione (Compound 23, Table III): Amixture of 7-acetamido-2-methylquinoline-5,8-dione (27) (prepared asdescribed in Example 27) (230 mg, 1 mmol), selenium dioxide (139 mg,1.25 mmol) in dry dioxane (3.5 mL) and water (0.13 mL) was stirred andrefluxed under argon for 9 hours. A fresh grade of 99.9+% of SeO₂(Aldrich) was used. Dioxane was purified and distilled according to theprocedure described in Perrin et al., Purification Of LaboratoryChemicals (Pergammon Press 1980). Dioxane (7 mL) was added, allowed toreflux for 10 min., and then filtered hot. The residue on the filterpaper was added to the reaction flask containing 20 mL ofdichloromethane, refluxed for 5 min and filtered. This process wasrepeated four more times. The filtrates were combined and evaporated togive 222 mg (yield of 91%) of a yellow solid which was7-acetamido-2-methylquinoline-5,8-dione (23). An analytical sampleobtained by sublimation (150-180EC/0.5 mm): mp 225EC dec.; IR (KBr)3346, 3085, 1721, 1687, 1652, 1609, 1504, 1328, 1216, 1124, 1068, 885cm⁻¹; ¹H NMR (CDCl₃)*2.33 (s, 3, COCH₃), 8.05 (s, 1, H-6), 8.31 (d, 1,J=8 Hz, H-3), 8.43 (br s, 1, NH), 8.62 (d, 1, J=8 Hz, H-4), 10.29 (s, 1,CHO); MS m/e (relative intensity) 244 (M+, 81), 216 (19), 202 (51), 175(37), 174 (21), 97 (19), 85 (20), 71 (30), 68 (18), 57 (34), 43 (100).Elemental analysis calculated for C₁₂H₈N₂O₄: C, 59.02; H, 3.30; N,11.47; Found: C, 58.99; H, 3.38; N, 10.91.

7-Chloroacetamido-2-Formylquinoline-5.8-Dione (Compound 24, Table III):In a 25 ml. round-bottomed flask equipped with a magnetic bar,water-cooled reflux condenser, and argon-filled balloon,7-chloroacetamido-2-methylquinoline-5,8-dione (28) (prepared asdescribed in Example 28) (0.529 g., 2 mmol), selenium dioxide (0.255 g.,2.3 mmol), 12 ml of dried, distilled 1,4-dioxane, and 0.25 ml. of waterwere stirred and slowly heated to reflux over a two-hour period. Thereaction was monitored by TLC and allowed to go to completion (29.5hours). The selenium was allowed to settle, and the supernatant solutionwas pipetted off and filtered. 1,4-Dioxane (10 ml.) was added to theresidue, stirred and refluxed for five minutes. The entire mixture wasfiltered and the selenium was washed with dichloromethane (10 ml.). Allfiltrates were combined and stored at 4EC overnight. Solid precipitatefrom the chilled mixture was filtered yielding 0.0985 g. of crudeproduct (yield of 18%). This was purified by recrystallization fromethyl acetate/dichloromethane. The original remaining filtrate wasdiluted with 50 ml of dichloromethane and washed with 3% sodiumbicarbonate solution (2×50 ml.). The aqueous layer was then extractedwith dichloromethane (2×20 ml.) The organic layers were dried withmagnesium sulfate and rotoevaporated to give a dark orange solid. Theproduct was dried under vacuum overnight and 0.2941 g. of product wasobtained (yield of 52%, total 70%). This product could be furtherpurified by flash chromatography and recrystallization from ethylacetate/dichloromethane: mp 190-192EC; ¹H NMR (CDCl₃)*4.26 (2H, s,C-7NHCOCH₂Cl), 8.04 (1H, s, C-6H), 8.33 (1H, d, J=8.1 Hz, C-3H), 8.62(1H, d, J=8.1 Hz, C-4H), 9.54 (1H, br. s, C-7NH), 10.28 (1H, s, C-2CHO);IR (KBr) v_(max) 3310, 3089, 2950, 2852, 1731, 1720, 1678, 1644, 1521,1118 cm⁻¹; EIMS, m/e (relative intensity) 278/280 (M+, 2.7/1, 95), 243(33), 229 (33), 215 (55), 202 (base), 175 (61), 146 (17); HRMS, m/e forC₁₄H₉ClN₂O₃ calculated 278.009435, found 278.008764; analysis forC₁₄H₉ClN₂O₃ calculated C, 51.72; H, 2.53; Cl, 12.72; N, 10.05; Found C,51.81; H, 2.9; Cl, 12.79; N. 9.86.

7-Butyramido-2-Formylquinoline-5,8-Dione (Compound 26, Table III): In a25 ml. round-bottomed flask equipped with a magnetic bar, water-cooledreflux condenser, and an argon filled balloon,7-butyramido-2-methylquinoline-5,8-dione (30) (prepared as described inExample 30) (0.516 g., 2 mmol), selenium dioxide (0.255 g., 2.3 mmol),12 ml. of dried, distilled 1,4-dioxane, and 0.25 ml. of water werestirred and slowly heated to reflux over a two-hour period. The reactionwas monitored by TLC and found to be complete after 33.5 hours. Theselenium metal was allowed to settle, and the supernatant solution waspipetted off and filtered. 1,4-Dioxane (10 ml.) was added to theresidue, stirred and refluxed for five minutes. The entire mixture wasfiltered and the selenium was washed with dichloromethane (10 ml.). Allfiltrates were combined and stored at 4EC overnight. This solution wasdiluted with 50 ml of dichloromethane and washed with a 3% sodiumbicarbonate solution (2×50 ml.). The organic layer was dried withmagnesium sulfate and rotoevaporated to give a pale yellow product. Thesolid was dried under vacuum overnight and weighed 0.356 g. (yield of65%). The product was recrystallized from ethyl acetate: mp. 208-210E C;¹H NMR (CDCl₃)*1.02 (3H, t, J=7.4 Hz, C-7NHCOCH₂CH₂CH₃), 1.70-1.89 (2H,m, 7NHCOCH₂CH₂CH₃), 2.52 (2H, t, J=7.4 Hz, 7NHCOCH₂CH₂CH₃), 8.06 (1H, s,C-6H), 8.31 (1H, d, J=8.0 Hz, C-3H), 8.39 (1H, br, s, C-7NH), 8.61 (1H,d, J=8.0 Hz, C-4H), 10.28 (1H, s, C-2CHO); IR (KBr) v_(max) 3299, 3081,2966, 2935, 2876, 1723, 1694, 1638, 1606, 1505 cm⁻¹; EIMS, m/e (relativeintensity) 272 (54), 202 (36), 175 (9); HRMS, m/e for C₁₄H₁₂N₂O₄calculated 272.079707, found 272.078696; analysis for C₁₄Ht₂N₂O₄calculated C, 61.76; H, 4.44; N, 10.29; found C, 61.31; H. 4.36; N,9.94.

TABLE III Elemental Compound R¹⁰ Hr. % Yield mp (° C.) ¹H, NMR (CDCl₃)MS Analysis or HRMS 23 CH₃ 9 91 225 (dec) 2.33(s, 3H), 8.05(s, 1H),8.31(d, 1H), 244 (M⁺, 81), 218 (19), Calc.: C, 59.02; H, 8.43(br s, 1H),8.62(d, 1H), 10.29(s, 202 (51), 175 (37), 174 3.30; N, 11.47; 1H) (21),97 (19), 85 (20), Found: C, 58.99; 71 (30), 68 (18), 57 (34), H, 3.38;N, 10.91 43 (base) 24 ClCH₂ 29.5 70 (crude) 190-192 4.26(s, 2H), 8.04(s,1H), 8.33(d, J= 278/280 (M⁺, 2.7/1.95), C₁₄H₉ClN₂O₃ 8.1Hz, 1H), 8.62(d,J=8.1Hz, 1H), 9.54 243 (33), 229 (33), 215 Calc. 278.009435 (br s, 1H),10.28(s, 1H) (55), 202 (base), 175 Found 278.008764 (61), 146 (17). 25(CH₃)₂CH 20 65 183-184 1.29(d, J=7Hz, 6H), 2.67-2.72(m, J= 272 (M⁺,Base), 229 6.92, 6.88, 6.96Hz, 1H), 8.06(s, 1H), (4.5), 217 (4.1), 2038.3(d, J=8Hz, 1H), 8.4(s, 1H), 8.6(d, (26.4), 175 (4.7) J=8Hz, 1H),10.29(s, 1H) 26 CH₃(CH₂)₂ 33.5 65 (crude) 208-210 1.02(t, J=7.4Hz, 3H),1.70-1.89(m, 272 (M⁺, 54), 202 (36), C₁₄H₁₂N₂O₄ 2H), 2.52(t, J=7.4Hz,2H), 8.06(s, 1H), 175 (9) Calc. 272.079707 8.31(d, J=8.0Hz, 1H), 8.39(brs, 1H), Found 272.078696 8.61(d, J=8.0Hz, 1H), 10.28(s, 1H) Calc. C:61.76, H: 4.44, N: 10.29 Found C: 61.31, H: 4.36, N: 9.94

Examples 27-30 Preparation of 7-Acetamido-2-Methylquinoline-5.8-diones

The 7-N-acyl-2-methylquinoline-5,8-diones used as starting materials inExamples 23-26 are compounds of formula M:

wherein

-   -   R¹⁰ is an alkyl or substituted alkyl,    -   R¹=H.    -   R²=H, and    -   R³=H.        They were prepared by one of two methods: (1) The oxidation of        the corresponding acylamido compounds with potassium dichromate        in glacial acetic acid as illustrated in Equation 4 above;        or (2) The Diels-Alder condensation of a 1-silyloxy-azadiane        with a 2-acetamido-6-bromobenzoquinone as illustrated in        Equation 2 above. Table IV lists four        7-N-acyl-2-methylquinoline-5,8-diones, their % yields, melting        points, elemental analyses, ¹H NMR and MS. The following are the        detailed procedures used for the preparation of these        quinolinediones.

7-Acetamido-2-Methylquinoline-5,8-Dione (Compound 27, Table IV):

This compound was prepared by the oxidation of the correspondingacylamido with potassium dichromate in glacial acetic acid. Into a 1000ml Erlenmeyer flask, 240 ml of glacial acetic acid, and5,7-diacetamido-2-methyl-8-acetoxyquinoline (31) (prepared as describedin Example 31) (6.3 g, 2.0 mmol) were added. To the resultingsuspension, a solution of potassium dichromate (17.64 g) in 200 ml ofwater was added. This black solution was magnetically stirred at roomtemperature for 20-24 hours. The solution was then poured into 900 ml ofwater and extracted with dichloromethane (5×200 ml). The organicextracts were washed with a solution of 5% sodium carbonate in asaturated salt solution (3×300 ml). The organic layer was driedovernight with anhydrous magnesium sulfate. The magnesium sulfate wasfiltered off, and the solvent was evaporated to leave an orange/yellowsolid. The solid 7-acetamino-2-methylquinoline-5,8-dione (27) was driedovernight under vacuum. 2.6 g of product was obtained for a yield of56%.

7-Acetamido-2-Methylquinoline-5.8-Dione (27):7-Acetamido-2-methylquinoline-5,8-dione (27) was also prepared byDiels-Alder condensation of a 1-silyloxy-azadiene with2-acetamido-6-bromobenzoquinone. The azadiene was prepared by thereaction of methyl vinyl ketone with t-butyldimethylsilyloxyamine in thepresence of molecular sieves as illustrated in Equation 2 above. In adry 50 mL round-bottomed flask containing 5.7 g of dry molecular sieve4AN, 1.4 g (0.02 mol) of freshly distilled methyl vinyl ketone in 10 mLdry dichloromethane was placed. Then a solution of theO(tert-butyldimethylsilyl)hydroxylamine (Aldrich) in 5 mL of drydichloromethane was added. The mixture was stirred at room temperatureunder argon for 48 h and then filtered off. Evaporation of the solutionunder reduced pressure and fractional distillation of the residueafforded 2.04 g (51% yield, bp 67-71° C./8 mm) of azadiene. Nuclearmagnetic resonance spectroscopy showed the product to be an E/Z mixture(7:3) which was used as such in the next reaction. An analytical sampleof the major isomer was obtained by silica gel column chromatographyusing petroleum ether and then petroleum ether plus ethylacetate (200:1)as solvents. IR (liquid film) 2959, 2931, 2860, 1630, 1581, 1462, 1363,1251, 1061, 983, 948, 871, 836, 808, 787, 674 cm⁻¹; ¹H NMR (CDCl₃)*0.15(s, 6, Si(CH₃)₂), 0.91 (s, 9, C(CH₃)₃), 1.95 (s, 3, C-2CH₃), 5.35-5.57(m, 2, =CH₂), 6.38-6.52 (m, 1, =CH); MS, m/e (relative intensity) 199(M⁺, 0.3), 142 (100), 75 (68), 68 (97), 42 (20); Elemental analysiscalculated for C₁₀H₂₁NOSi: C, 60.24; H, 10.62; N, 7.03; found: C, 60.22;H, 10.56; N, 7.10.

Then, 7-acetamido-2-methylquinoline-5,8-dione (27) was prepared. Asolution of bromoquinone (317 mg, 1.3 mmol),1-(tert-butyldimethylsilyloxy)-2-methyl-1-aza-1,3-butadiene (130 mg,0.65 mmol) in 28 mL dry chlorobenzene was refluxed under argon for 22 h.Chlorobenzene (10 mL) was added and refluxed for another 2 h. Thereaction mixture was allowed to cool and then put onto a silica gelcolumn (2×9.5 cm). The column was eluted with ethylacetate/petroleumether (2:1), EtoAc and then EtOH. The solvent was removed, benzene wasadded, heated and filtered. Evaporation of the filtrate gave 99 mg ofyellow solid (27) (a yield of 66%). An analytical sample recrystallizedfrom chloroform gave mp 217° C. (dec.); IR (KBr) 3339, 1750, 1714, 1679,1651, 1609, 1588, 1510, 1370, 1314, 1222, 1131, 744, 519 cm⁻¹; ¹H NMR(CDCl₃)*2.30 (s, 3, COCH₃), 2.75 (s, 3, ArCH₃), 7.55 (d, 1, J=8 Hz,H-3), 7.909 (s, 1, H-6), 8.29 (d, 1, J=8 Hz, H-4), 8.38 (br s, 1, NH);MS m/e (relative intensity) 230 (M+, 69), 188 (81), 161 (100), 132 (20),93 (15), 43 (31). Analysis calculated for C₁₂H₁₀N₂O₃: C, 62.61; H, 4.38;N, 12.17; Found: C, 62.52; H, 4.28; N, 11.93.

7-Chloroacetamido-2-Methylquinoline-5,8-Dione (Compound 28, Table IV):This compound was prepared by the oxidation of the correspondingacylamido with potassium dichromate in glacial acetic acid. In a 500 ml.round-bottom flask equipped with a magnetic bar,5,7-bis(chloroacetamido)-8-hydroxy-2-methylquinoline (32) (prepared asdescribed in Example 32) (3.42 g., 0.01 mol) was suspended in 122 ml. ofglacial acetic acid. A solution of potassium dichromate (8.8 g., 0.03mol) in 115 ml. of water was added and the resulting dark solution wasstirred at room temperature overnight. The solution was extracted withdichloromethane (12×50 ml.). The organic extracts were washed with 3%sodium bicarbonate solution (200 ml.), dried with magnesium sulfate, androtoevaporated to yield a bright yellow solid. After vacuum drying, thenearly pure product weighed 1.56 g. (yield of 59%). The product wasrecrystallized from ethyl acetate: mp 196-200EC (dec.); ¹H NMR(CDCl₃)*2.76 (3H, s, C-2CH₃), 4.23 (2H, s, C-7NHCOCH₂Cl), 7.56 (1H, d,J=8.1 Hz, C-3H), 7.89 (1H, s, C-6H), 8.30 (1H, d, J=8.1 Hz, C4H), 9.48(1H, br. s, C-7NH); IR (KBr) v_(max) 3299, 3078, 2948, 1712, 1683, 1641,1614, 1588, 1515, 1398, 1384, 1323, 1130 cm⁻¹; EIMS, m/e (relativeintensity) 264/266 (M⁺, 2.9/1, 67) 229 (62), 215 (74), 201 (43), 188(86), 161 (base), 132 (21); HRMS, m/e for C₁₄H₉ClN₂O₃, calculated264.030170, found 264.029824; analysis for C₁₄H₉ClN₂O₃ calculated: C,54.46; H, 3.43; Cl, 13.40; N, 10.58; found: C, 54.19; H, 3.37; Cl,13.29; N, 10.36.

7-Butyramido-2-Methylquinoline-5.8-Dione (Compound 30. Table IV): Thiscompound was prepared by the oxidation of the corresponding acylamidowith potassium dichromate in glacial acetic acid. In a 500 ml.round-bottomed flask equipped with a magnetic bar,5,7-dibutyramido-8-butyroxy-2-methylquinoline (34) (prepared asdescribed in Examples 31-34) (3.29 g., 8.25 mmol) was suspended in 122ml of glacial acetic acid. A solution of potassium dichromate (8.8 g.,0.03 mol) in 115 ml. of water was added and stirred. The stirredsuspension began to dissolve but, after two hours, more solid continuedto precipitate. Dichloromethane (70 ml.) was added to promote solution.The resulting two-phase solution mixture was stirred at room temperatureovernight. The two-phase solution mixture was extracted withdichloromethane (12×50 ml.). The organic extracts were washed with a 3%sodium bicarbonate solution (200 ml.), dried with magnesium sulfate, androtoevaporated to yield an orange-yellow solid. After vacuum drying, thesolid weighed 1.65 g. (a yield of 77%). The product was recrystallizedfrom ethyl acetate: mp. 188-189EC; ¹H NMR (CDCl₃) 1.00 (3H, t, J=7.4 Hz,C-7NHCOCH₂CH₂CH₃), 1.69-1.82 (2H, m, C-7NHCOCH₂CH₂CH₃), 2.48 (2H, t,J=7.4 Hz, C-7NHCOCH₂CH₂CH₃), 2.74 (3H, s, C-2CH₃), 7.53 (1H, d, J=8.0Hz, C-3H), 7.90 (1H, s, C-6H), 8.28 (1H, d, J=8.0 Hz, C-4H), 8.36 (1H,br. s, C-7NH); IR (KBr) v_(max) 3338, 3275, 2964, 2935, 2875, 1711,1682, 1654, 1644, 1615, 1588, 1502, 1323, 1310, 1133 cm⁻¹; EIMS, m/e(relative intensity) 258 (81), 215 (7), 188 (base), 161 (66); HRMS, m/efor C₁₄H₁₄N₂O₃ calculated 258.100442, found 258.100227; analysis forC₁₄H₁₄N₂O₃ calculated C, 65.11; H, 5.46; N, 10.85; found C, 65.22; H.5.51; N 10.01.

TABLE IV Elemental Compound R¹⁰ % Yield mp (EC) ¹H, NMR (CDCl₃) MSAnalysis or HRMS 27 CH₃ 66 217 2.30(s, 3H), 2.75(s, 3H), 7.55(d, 1H),7.90 230 (M⁺, 69), 188 (81), Calc. C, 62.61; H, 4.38; (s, 1H), 8.29(d,1H), 8.38(br s, 1H) 161 (100), 132 (20), 93 N, 12.17 (15), 43 (31) FoundC, 62.52; H, 4.28; N, 11.93 28 ClCH₂ 59 196-200 (dec) 2.76(s, 3H),4.23(s, 2H), 7.56(d, J= 264/266 (M⁺2.9/1, 67), C₁₄H₉ClN₂O₃ 8.1Hz, 1H),7.89(s, 1H), 8.30(d, J=8.1Hz, 229 (62), 215 (74), 201 Calc: 264.0301701H), 9.48(br s, 1H). (43), 188 (86), 161 Found: 254.029824 (base), 132(21) Calc.: C, 54.46; H, 3.43; N, 10.58; Cl, 13.40 Found: C, 54.19, H,3.37, N, 10.36; Cl, 13.29 29 (CH₃)₂CH 73 189-190 (dec) 1.26(d, J=6.76Hz,6H), 2.7(m, J=6.76Hz, 258 (M⁺, base), 215 1H), 2.75(s, 3H), 7.54(d,J=8Hz, 1H), 7.9 (25.9), 189 (41.5), 161 (s, 1H), 8.29(d, J=8Hz, 1H),8.42(br s, (266), 108 (41.1) 1H) 30 CH₃(CH₂)₂ 77 188-189 1.00(t,J=7.4Hz, 3H), 1.69-1.82(m, 2H), 258 (M⁺, 81), 215 (7), C₁₄H₁₄N₂O₃2.48(t, J=7.4Hz, 2H), 2.74(s, 3H), 7.53(d, 188 (base), 161 (66) Calc258.100442 J=8.0Hz, 1H), 7.90(s, 1H), 8.28(d, J= Found 258.100227 8.0Hz,1H), 8.36(br s, 1H) Calc. C, 65.11; H, 5.46; N, 10.85 Found: C, 65.22;H, 5.51; N, 10.91

Examples 31-34 Preparation of5,7-Diacylamido-8-Acetoxy-2-Methylquinolines

These compounds, which were used as starting materials for thepreparation of the 7-N-acyl-2-methyl-quinoline-5,8-diones of Examples27-30, were prepared by the reduction of5,7-dinitro-8-hydroxy-2-methylquinoline by molecular hydrogen in thepresence of palladium on charcoal (Pd/C) followed by treatment with thedesired anhydrides as illustrated in Equation 5 above. Table V listsfour 2-methylquinolines prepared by this method, their % yields, meltingpoints, elemental analysis, ¹H NMR and MS. The following procedures wereused to prepare these compounds. Also, the preparation of5,7-dinitro-8-hydroxy-2-methylquinoline is described; this compound wasprepared according to a literature method.

Preparation of 5.7-Diacetamido-2-Methyl-8-Acetoxyquinoline (Compound 31,Table V): Into a 500 ml hydrogenation bottle,5,7-dinitro-8-hydroxy-2-methylquinoline (47) (preparation describedbelow) (6.03 g, 24.2 mmol), water (100 ml), and concentrated HCl (13 ml)were added. To this suspension, 5% Pd/C (2.00 g) was added as acatalyst. This mixture was hydrogenated at 40 psi overnight or until nomore hydrogen is absorbed. This reaction takes approximately four hours.

The resultant solution was then carefully vacuum filtered to remove thePd/C. Note: Do not rinse the Pd/C that is on the filter paper withwater. This will dilute the solution which is concentration-sensitive.The filtrate should take up, at most, 130 ml.

The filtrate was transferred to a 500 ml beaker. To the filtrate, sodiumsulfite (2.85 g), sodium acetate (3.58 g), and acetic anhydride (2.5 ml)were added with stirring using a magnetic stirrer. Generally, noprecipitate formed. After 30 minutes, the same amounts of the abovethree compounds were added again. The addition of acetic anhydride (7.5ml) caused an orange/white precipitate to form. Note: If no precipitateforms, remove the solvent under reduced pressure and heat until a smallamount of solution is obtained. Add the three compounds (sodium sulfite,sodium acetate, and acetic anhydride) in small quantities (approximatelyone-quarter of that required in the procedure), and then wait 15minutes. Next add 5 ml acetic anhydride and stir the mixture vigorously.Add small amounts of sodium sulfite and sodium acetate while vigorouslystirring for 10 minutes. Stop the stirring and allow the mixture tostand, the precipitate should then form. This problem can be avoided,however, by following the prescribed procedure exactly as written. Themixture was then vacuum filtered, and the precipitate was washed threetimes with water (3×100 ml). This process washed the orange color out ofthe precipitate, and left a white solid on the filter paper. Thefiltrate was evaporated down to one-quarter of its volume. Anyprecipitate that formed was filtered and washed, as above. The filtratewas treated with sodium acetate and acetic anhydride again until no moreprecipitate was formed (Note: The addition of the acetic anhydride iswhat generally causes more precipitate to form). All washed precipitateswere combined and allowed to dry overnight under the hood or on a vacuumpump. The yield was 80% (6.05 g of product, mp 255EC).

5.7-Bis(Chloroacetamido)-8-Hydroxy-2-Methylquinoline (Compound 32. TableV): In a 500 ml heavy-walled hydrogenation bottle,8-hydroxy-2-methyl-5,7-dinitroquinoline (47) (5.25 g., 0.021 mol) and 5%Pd/C (1.75 g.) were suspended in 90 ml. of water and 9 ml. ofconcentrated HCl. This mixture was shaken under 30 psi of hydrogen for20 hours. The catalyst was filtered off, and the dark red solutioncontaining the dihydrochloride salt of5,7-diamino-8-hydroxy-2-methylquinoline was placed in a 250 ml.rounded-bottomed flask equipped with a magnetic bar. To this stirredsolution was added in sequence as quickly as possible, sodium sulfite(12 g.), sodium acetate (16 g.), and chloroacetic anhydride (65 g.).Heat was evolved, and the formation of a light colored, thin precipitateoccurred. After 15 minutes of stirring the precipitate dissolved. Anadditional amount of chloroacetic anhydride (5 g.) was added and thereaction mixture stirred for one hour. The solution was concentratedunder reduced pressure until a precipitate appeared. This mixture waspoured into a 400 ml beaker containing 100 ml. of ice-water and stirredfor 5 minutes. The mixture was vacuum filtered and the product waswashed with cold ethanol, dried under vacuum and collected (2.14 g.).The filtrate, upon standing overnight, yielded an additional amount ofproduct (1.47 g.). Total weight of the product was 3.61 g. (a yield of50%). The product was recrystallized from ethanol and water: mp 194-196°C. (dec.); ¹H NMR (DMSO-d₆)*2.71 (3H, s, C-2CH₃), 4.37 (2H, s, COCH₂Cl),4.44 (2H, s, COCH₂Cl), 7.43 (1H, d, J=8.8 Hz, C-3H), 8.15 (1H, d, J=8.8Hz, C-4H), 8.16 (1H, s, C-6H), 9.89 (1H, br s, NH), 10.18 (1H, br s,NH); IR (KBr) v_(max) 3392, 3361, 3262, 3005, 2950, 1680, 1660, 1646,1557, 1523, 1508, 1456, 1416 cm⁻¹; EIMS, m/e (relative intensity)341/343 (M+, 1.5/1, 52), 305 (60), 292 (base), 264 (28), 250 (7), 228(60), 214 (10), 200 (12), 188 (70), 173 (11), 160 (17); HRMS, m/e forC₁₄H₁₃Cl₂N₃O₃ calculated 341.033397, found 341.033888; analysis forC₁₂H₁₃Cl₂N₃O₃, calculated C, 49.14; H, 3.83; Cl, 20.72; N, 12.28; found,C, 49.24; H, 3.89; Cl, 20.43; N, 12.16.

5.7-Dibutyramido-8-Butyroxy-2-Methylquinoline (Compound 34, Table V): Ina 500 ml heavy-walled hydrogenation bottle,8-hydroxy-2-methyl-5,7-dinitroquinoline (47) (5.0 g., 0.02 mol) and 5%Pd/C (1.5 g.) were suspended in 100 ml. of water and 12 ml. ofconcentrated HCl. This mixture was shaken under 30 psi of hydrogen for15 hours. The catalyst was filtered off, and the dark red solutioncontaining the dihydrochloride salt of5,7-diamino-8-hydroxy-2-methylquinoline was placed in a 250 ml.round-bottomed flask equipped with a magnetic bar. To this stirredsolution, was added in sequence as quickly as possible, sodium sulfite(12 g.), sodium acetate (16 g.), and butyric anhydride (65 ml.). Thethick whitish precipitate which continued to form over a 3-hour periodwas filtered and washed with water. After vacuum drying, the productweighed 7.4 g (a yield of 93%). Since recrystallization withmethanol-water resulted in the hydrolysis of the butyrate, analyses wereperformed on the crude product: mp. 195-205EC (dec.); ¹H NMR(DMSO-d₆)*0.92 (3H, t, J=8.0 Hz, NHCOCH₂CH₂CH₃), 0.96 (3H, t, J=8.0 Hz,NHCOCH₂CH₂CH₃), 1.08 (3H, t, J=8.0 Hz, OCOCH₂CH₂CH₃), 1.52-1.72 (4H, m,2 NHCOCH₂CH CH₃), 1.73-1.89 (2H, m, OCOCH₂CH CH₃), 2.30-2.40 (4H, m,2NHCOCH₂CH₂CH₃), 2.58 (3H, s, C-2CH₃), 2.70 (2H, t, J=8.0 Hz,OCOCH₂CH₂CH₃), 7.37 (1H, d, J=8.8 Hz, C-3H), 8.21 (1H, d, J=8.8 Hz,C-4H), 8.24 (1H, s, C-6H), 9.65 (1H, br s, C-5NH), 9.94 (1H, br s,C-7NH); IR (KBr) v_(max) 3343, 3258, 2964, 2935, 2874, 1738, 1693, 1656,1630, 1541, 1505 cm⁻¹.

8-Hydroxy-2-Methyl-5.7-Dinitroquinoline (Compound 47): Compound 47 wasprepared according to the method described in Boger et al., J. Org. Chem50, 5782 (1985). In a 500 ml. Erlenmeyer flask, equipped with a magneticbar was placed 300 ml. of a 70% (v/v) solution of concentrated nitricacid-sulfuric acid. The solution was stirred and cooled in an ice bath.To this stirred, chilled solution, 8-hydroxy-2-methylquinoline (30.0 g.,0.188 mol) was added in small portions over a ten minute period. Uponaddition of the 8-hydroxy-2-methylquinoline, a brownish gas was evolved.The mixture was continually stirred in the ice bath for two hours. Themixture was then poured into a 2 L. beaker containing 1200 ml. ice water(1:2, ice:water) and stirred vigorously with a glass rod. The brightyellow precipitate was vacuum filtered, washed with ethanol (300 ml.),and then washed with ethyl ether (2×300 ml.). The resulting8-hydroxy-2-methyl-5,7-dinitroquinoline was left to air dry overnightand then further dried under vacuum for 24 hours. The yellow solidweighed 28.1 g. (a yield of 60%): mp. 295-300° C.; ¹H NMR (DMSO-d₆)*2.93(3H, s, C-2CH₃), 8.13 (1H, d, J=9.1 Hz, C-3H), 9.20 (1H, s, C-6H), 9.65(1H, d, J=9.1 Hz, C4H); IR (KBr) v_(max) 3067, 1593, 1527, 1336, 1320,1299, 1259 cm⁻¹.

TABLE V Elemental Compd R¹⁰ % Yield mpN (C) ¹H NMR (DMSO) MS Analysis orHRMS 31 CH₃ 80 284 (dec) 2.14(s, 3H), 2.15(s, 3H), 2.41(s, 3H), 2.60Calc. C-60.94, H-5.43, (s, 3H), 7.37(d, 1H), 8.24(d, 1H), 8.31(s,N-13.33 1H), 9.75(s, 1H), 10.02(s, 1H) Found C-61.15, H-5.39, N-13.27 32ClCH₂ 50 194-196 (dec) 2.71(s, 3H), 4.37(s, 2H), 4.44(s, 2H), 7.43341/343 (M⁺ 1.5/1, 52), C₁₄H₁₃Cl₂N₃O₃ (d, J=8.8Hz, 1H), 8.15(d J=8.8Hz,1H), 305 (60), 292 (base), 264 Calc 341.033397 8.16(s, 1H), 9.89(br s,1H), 10.18(br s, (28), 250 (7), 228 (60), Found 341.033888 1H) 214 (10),200 (12), 188 (70), 173 (11), 160 (17) 33 (CH₃)₂CH 75 (crude) 245-247(dec) 1.12(d, J=6.8Hz, 6H), 1.19(d, J=6.8Hz, 399 (M⁺, 0.2), 329 6H),1.38(d, J=7Hz, 6H), 2.6(s, 3H), (base), 286 (67.9), 259 2.84-2.72(m,2H), 3.04-3.01(m, 1H), (39.4), 241 (12.9), 188 7.4(d, J=8.8Hz, 1H),8.13(s, 1H), 8.23 (75.2) (d, J=8.8Hz, 1H), 9.62(br s, 1H), 9.92 (br s,1H) 34 CH₃(CH₂)₂ 93 (crude) 200-208 (dec) 0.73(t, J=7.0Hz, 3H), 0.76(t,J=7.0Hz, 329 (95), 286 (20), 259 C₁₈H₂₃N₃O₃ 3H), 1.33-1.55(m, 4H),2.1-2.3(m, 4H), (98), 241 (17), 216 (6), Calc. 329.173942 2.3(s, 3H),7.17(d, J=8.8Hz, 1H), 7.82(s, 188 (base), 161 (13) Found 329.173043 1H),7.90(d, J=8.8Hz, 1H), 9.28(br s, 1H), 9.52(br s, 1H)

Examples 35-46 Preparation of Tryptophan Analogs

The tryptophan analogs used for the synthesis of lavendamycin analogs1-22 are listed in Table VI. They have formula L:

wherein R⁵ ₅ R⁷, R⁸ and R⁹ are H, and R⁴, R⁶ and Y are as given in TableVI below. Tryptophan esters 36, 37, and 44-46 (see Table VI) weresynthesized as illustrated in Equation 6 above. Tryptophan esters 40-41(see Table VI) were synthesized as illustrated in Equation 7 above. Theprocedures used for the synthesis of all of the tryptophan analogs aredescribed below.

Tryptorphan Esters 36, 37 and 44-46 (Table VI): Tryptophan (or^(b)-methyltryptophan) (3 g, 0.015 mol) and dry HCl-saturated isoamylalcohol (or other alcohols) (180 ml) were stirred and refluxed for 24hr. The reaction mixture was rotoevaporated to give a white crystalsalt. After it was washed with ether, the salt was neutralized with 14%NH₄OH in 25 ml ethyl acetate. Then, it was washed with water and driedwith anhydrous magnesium sulfate. Finally, it was rotoevaporated to give33.21 g colorless oil (or crystal) (yield 80-90%).

Tryptophan Esters 40 and 41 (Table VI):

(1) Carbobenzoxy-tryptophan (or Carbobenzoxy-^(b)-Methyltryptophan): Amixture of tryptophan (4.1 g, 0.02 mol) (or b-methyltryptophan), NaOH(2N, 10 ml), water (20 ml), benzylchloroformate (3.4 g., 0.02 mol, in2.2 ml of toluene) was stirred in an ice bath. Another 5 ml of NaOH (4N)was added dropwise over 20 minutes and stirred for an additional 10minutes. The mixture was acidified to Congo Red with hydrochloric acid,filtered, washed with cold water and dried to give 5.86 g. product(yield 87%). N-CBZ-^(b)-methyltryptophan was produced in a yield of 93%.

(2) N-CBZ-Tryptophan (or N-CBZ-^(b)-Methyltryptophan)N,N-Dimethylethylamino Ethyl Ester: N-CBZ-tryptophan (3.384 g., 0.01mol) (or N-CBZ-^(b)-methyltryptophan), N,N-dimethylaminoethylchloride(2.1 N benzene solution, 6 ml), ethyl acetate (20 ml) was stirred andheated. Triethylamine (1.4 ml) was added dropwise, and the mixture wasthen refluxed 5 hr. and filtered. The filtrate was washed with saturatedNaCl in water, 5% NaHCO₃ and saturated NaCl in water, dried withanhydrous MgSO₄, rotoevaporated to give 1.76 g. of a spongy gel (a yieldof 43%). N-CBZ-^(b)-methyltryptophan-N,N-dimethylethylaminoethyl esterwas produced in a yield of 81%.

(3) Tryptophan (or ^(b)-Methyltryptophan) N,N-Dimethylethylamino EthylEster: A mixture of ammonium formate (125 mg) andN-CBZ-tryptophan-N,N-dimethylethylaminoethyl ester (205 mg, 0.5 mmol)(or N-CBZ-^(b)-methyltryptophan-N,N-dimethylethylaminoethyl ester), DMF(5 ml), 10% Pd/C (100 mg) was stirred under argon at room temperaturefor 30 minutes and then filtered. The filtrate was rotoevaporated to anoil. Chloroform (10 ml) was added to the oil and filtered again. Thefiltrate was rotoevaporated to dryness and dissolved in ethyl acetate(25 ml), washed with 3×5 ml saturated NaCl solution, dried overanhydrous MgSO₄, evaporated in a vacuum to give 101 mg of the oilyproduct (a yield of 74%). ^(b)-methyltryptophan N,N-dimethylethylaminoethyl ester was produced in a yield of 68%.

Tryptophan Analogs 35, 38, 42 and 43 (Table VI): These compounds arecommercially available as the hydrochloride salts. They were convertedto the free amines by neutralization with 14% ammonium hydroxide,followed by extraction with ethyl acetate.

Tryptophan Ester 39 (Table VI): This compound was made as described inBehforouz et al., J. Heterocycl. Chem., 25, 1627 (1988).

TABLE VI Compound R⁴ R⁶ Y 35 H H CO₂CH₃ 36 CH₃ H CO₂CH₂CH₂CH(CH₃)₂ 37 HH CO₂CH₂CH₂CH(CH₃)₂ 38 H H CO₂(CH₂)₇CH₃ 39 CH₃ H CO₂CH₃ 40 H HCO₂CH₂CH₂N(CH₃)₂ 41 CH₃ H CO₂CH₂CH₂N(CH₃)₂ 42 H H CO₂(CH₂)₃CH₃ 43 H HCO₂NH₂ 44 H H CH₂—CH₂ CO₂CH  CH₂ CH₂—CH₂ 45 H OCH₃ CO₂(CH₂)₃CH₃ 46 H FCO₂(CH₂)₃CH₃

Example 47 In Vitro Cytotoxicity

The in vitro cytotoxicity of some of the lavendamycin analogs wasdetermined according to the methods described below against a panel offive cell types. A few quinones were also tested for cytotoxicity.

A. Cell Lines and Culture Conditions. Stock cultures of normal ratkidney epithelial (NRKE) cells [De Larco et al., J. Cell Physiol., 94,335 (1978)1 and Lewis lung carcinoma cells [Suguira et al., Cancer(Phila.), 5, 382 (1952)] were obtained from the American Type CultureCollection (ATCC, Rockville, Md.). Both cell lines were grown inantibiotic-free culture medium [Dulbecco's high glucose MEM supplementedwith 10% fetal bovine serum]. The cell number of the Lewis lung and NRKEcell lines was expanded by 5 passages, and the cell lines were stored inliquid nitrogen, all according to standard procedures [Freshney, Cultureof Animal Cells: A Manual of Basic Technique, pages 6, 216-225 (2nd ed.1987)]. These cryopreserved cells were used for the assays describedbelow. All cell lines were grown using standard cell culture methods.[Freshney, Culture Of Animal Cells: A Manual Of Basic Technique, page127 (2nd ed. 1987)] All cell culture manipulations were done under goldfluorescent light to prevent damage by photooxidation.

B. Selection of Transformed Cells and Oncogene Transfections. NRKE cellstransformed by the ras^(K), ras^(N), and ras^(H) oncogenes were used inthe panel. Only transformants with a minimal deviation phenotype ascompared to NRKE cells were used. Transformants with this phenotype havea low oncogene copy number and in vitro growth characteristics the sameas nontransformed NRKE cells. These criteria were chosen so thatcompounds were evaluated for antitumor activity, rather than anti-growthactivity based on the rate of cell division.

The ras oncogene-transformed NRKE cells were prepared as follows. Theplasmids pUCEJ6.6 (no. 41028), containing a transforming human ras^(H)gene, pNRsac (no. 41031) containing a transforming human ras^(N) gene,pKSma (no. 41048), containing a v-ras^(K) gene, and the RSVneo gene wereobtained from the ATCC. NRKE cells were cotransfected with an oncogeneplus the RSVneo gene using standard calcium phosphate coprecipitationand neomycin resistance selection methods. [Davis et al., Basic MethodsIn Molecular Biology, 285 (1985)]. Approximately 14 days aftertransfection and selection with G418 antibiotic (Sigma Chemical Co., St.Louis, Mo.), colonies were isolated by the ring cloning procedure.[Freshney, Culture Of Animal Cells: A Manual Of Basic Technique (2nd ed.1987)]. The clones were stored in liquid nitrogen after expansion oftheir cell number by 4 passages in culture medium with G418.Approximately 50 clones were evaluated by several criteria for their usein the in vitro cytotoxicity and in vivo antitumor tests describedbelow. The criteria used to select clones were the following: (1) theclone had to have the same growth rate as the parental NRKE cells, (2)the growth rate of the clone must be stable with repeated passage (e.g.,up to 100 subcultures), and (3) they must be tumorigenic inimmunologically deficient mice. Criteria 1 and 2 are key to assuring thedifferential cytotoxicity assay selects for compounds that haveantitumor activity rather than a general anti-growth activity. Oncogenecopy number was determined using standard Southern blotting procedures[Current Protocols in Molecular Biology, Chapter 2, Frederick M. Ausuhelet al. eds., 1987-1992]. Cell growth characteristics were evaluatedusing standard methods (Freshney, Culture of Animal Cells: A Manual ofBasic Technique, pages 227-244 (2nd ed., 1987)].

The clones K/1-NRKE, transformed by v-ras^(K) gene, H/1.2-NRKE,transformed by the human ras^(H) gene, and N/4.2-NRKE transformed by thehuman ras^(N) gene, were selected for in vitro cytotoxicity and in vivoantitumor testing. These clones have 3 to 4 oncogene copies that arestably integrated. The in vitro doubling time of these cells is 24hours, which is the same as NRKE cells.

C. In Vitro Cytotoxicity Assay. Compounds were screened for antitumoractivity with an oncogene-based differential cytotoxicity assay. Withthis assay the cytotoxic action of compounds againstoncogene-transformed cells relative to their action against thenontransformed parent epithelial cells was determined. For theevaluation of compounds, the Lewis lung carcinoma was used as a murinereference tumor. This tumor is commonly used in screens for newoncolytics. The differential cytotoxicity assay can identify compoundsthat interact directly with oncogene proteins, but more importantly itcan also find compounds that interfere with the biochemical pathwaysactivated or driven by oncogenes. The inhibitory action in both casesmay be specific for a transformed cell.

Briefly, the procedure used for the differential cytotoxicity assay isas follows. Cell suspensions were prepared by trypsin dissociation usingstandard methods [Freshney, Culture Of Animal Cells: A Manual Of BasicTechnique, page 1321, and 50 cells were seeded into each well of 12-wellculture dishes. Groups of triplicate wells were divided into mediacontrol, drug vehicle-control and drug treatment groups. One day afterseeding, media was replaced with media containing vehicle or drugs, andthe cultures were incubated for an additional 5 days.

After exposure to the vehicles or drugs, the cultures were washed, fixedand stained with a modification of Mallory's stain. [Richardson et al.,Stain Technol., 35, 313 (1960)]. Colony number and colony area weredetermined with an Artek model 982 image analyzer (Artek System Corp.,Farmington, N.Y.). The cytocidal action of compounds was determined fromthe colony number as originally described by Puck and Marcus, J. Exp.Med., 103, 653 (1956). Colony areas were normalized for colony numberand were used to determine if a compound had a cytostatic action. Theconcentration giving 50% cell kill (LC₅₀) was determined, and adifferential index of cytotoxicity was determined by dividing the LC₅₀value for the normal epithelial cells by the LC₅₀ value for the tumorcells. The differential index value was used to determine the amount ofselective toxicity a compound had for the tumor cells. All data analysiswas performed using SAS software [SAS Users Guide: Statistics (Version 5edition, SAS Institute Inc., Cary, N.C. 1985)] on a VAX 8300 computer.The results are shown in Tables VII, VIII and IX.

TABLE VII LC₅₀ (:M) Compound NRKE K/1 H/1.2 3LL 27; R¹⁵ = NHAc R¹, R³ =H, R¹⁶ = CH₃ 2.4 5.9 4.4 2.0 23; R¹⁵ = NHAc R¹, R³ = H, R¹⁶ = CHO 1.32.6 2.5 1.2 48; R¹⁵ = NHAc R¹, R³ = H, R¹⁶ = COOH >33 >33 31 26 49; R¹⁵= NHAc R¹ = H, R³ = CH₃, 1.3 1.8 1.1 1 R¹⁶ = H 50; R¹⁵ = NH₂ R¹ = H, R³= CH₃, 0.8 1.3 0.6 0.8 R¹⁶ = H 51; R¹⁵ = NH₂ R¹, R³ = H, R¹⁶ = CHO 2.44.3 2.2 2.3 52; R¹⁵ = H, R¹ = NHAc, R³ = H, 21.5 >33 20 >33 R¹⁶ = CH₃53; R¹⁵ = OCH₃, R¹, R³ = H, R¹⁶ = CH₃ 6.9 11.8 4.2 7.4 54; R¹⁵ =O(CH₂)₄OH, R¹, R³ = H, 10.5 13.5 10.5 3.8 R¹⁶ = CH₃ 1; R¹⁵ = NHAc, R¹,R³ = H, R¹⁶ = BC 0.9 0.1 0.7 >33 55; R¹⁵ = NH₂, R¹, R³ = H, R¹⁶ = BC 0.10.2 0.1 0.5 NRKE = Normal Rat Kidney Epithelial Cells. K/1 = ras ^(K)Transformed NRKE Cells. H/1.2 = ras ^(H) Transformed NRKE Cells. 3LL =Lewis Lung Carcinoma Cells.

BC=^(b)-carboline moiety having the formula:

In Table VII, the compounds tested have the following formula:

Compounds 1, 23 and 27 were prepared as described in Examples 1, 23 and27. Compounds 49 and 52 were prepared according to Equation 2. Compound48 was obtained by the oxidation of 23. Treatment of 27 with methanol inthe presence of acid gave 53, and treatment of 27 with tetrahydrofuranin the presence of acid gave 54. Compounds 50, 51 and 55 were obtainedby the acid hydrolysis of 49, 23 and 1 respectively.

In Table VIII and IX, the compounds tested have the following formula:

TABLE VIII Substituent LC50 (M) Compound R¹³ R¹¹ R⁴ NRKE H/1.2 K/1 N/4.23LL 1 NHAc CH₃ CH₃ 0.90 0.70 0.10 N.T. >33.00 17 NHAc CH₂ H 0.50 0.510.11 0.52 0.84 18 NHAc CH₂CH₂CH(CH₃)₂ CH₃ 4.61 4.30 5.00 3.89 7.40 19NHAc CH₂CH₂N(CH₃)₂ CH₃ 0.09 0.09 0.06 0.09 0.48 20 NHAc CH₂CH₂CH(CH₃)₂ H1.62 1.42 0.09 1.50 1.69 21 NHAc (CH₂)₇CH₃ H >33.00 7.60 0.259.00 >33.00 22 NHAc CH₂CH₂N(CH₃)₂ H 0.37 0.24 0.14 0.29 0.50 55 NH₂ CH₃CH₃ 0.1 0.1 0.2 N.T. 0.5

TABLE IX Substituent Differential Index Compound R¹³ R¹¹ R⁶ H/1.2 K/1N/4.2 3LL 1 NHAc CH₃ CH₃ 1.29 9.00 — >0.03 17 NHAc CH₃ H 0.98 4.55 0.960.59 18 NHAc CH₂CH₂CH(CH₃)₂ CH₃ 1.07 0.92 1.19 0.62 19 NHAcCH₂CH₂N(CH₃)₂ CH₃ 1.00 1.50 1.00 0.19 20 NHAc CH₂CH₂CH(CH₃)₂ H 0.6218.00 1.08 0.96 21 NHAc (CH₂)₇CH₃ H >4.3 >132.00 >3.66 −1.00 22 NHAcCH₂CH₂N(CH₃)₂ H 1.54 2.64 1.29 0.74 55 NH₂ CH₃ CH₃ 1.0 0.50 — 0.20 N.T.= Not tested N/4.2 = ras^(K) transformed NRK cells See Table VII for NRKK/1, H/1.2, 3LL and Ac

As shown in Table VIII, the lavendamycin analogs 1 and 17-22 were alltoxic to one or more of the tumor cells tested. The lavendamycin analogsalso generally showed selective cytotoxicity against the ras oncogenebearing cells (see Tables VII and IX). In particular, lavendamycinanalogs 1, 20, and 21 showed unprecedented highly selective toxicity(9-, 18-, and 132-fold, respectively) against ras^(K) oncogene bearingtumor cells as compared to the normal, non-transformed cells. Bycomparison, the prior art compound lavendamycin methyl ester 55 has aselective toxicity of 0.5 (Table IX).

Example 48 In Vivo Activity Against Tumors

The antitumor activity of N-acetyl-lavendamycin methyl ester (1),N-acetyldemethyllavendamycin isoamyl and n-octyl esters (20 and 21) andcyclophosphamide (CY) (reference) were evaluated against K/1-NRKE tumor(ras^(K)-transformed NRKE cells) grown as a xenograft in nude mice.

1. The Mice

Female CDl nu/nu nude and female C57B1/6 mice were obtained from CharlesRivers, Inc. Animals were housed in plastic shoebox type cages coveredwith a microisolator top. All animal feed, water, bedding and cages weresterilized. The animals were provided water (pH 3.0) and Purina mousediet ad libitum. All animal manipulations were done with sterileprocedures in a 100% exhaust, vertical laminar flow, HEPA filtered hood.To maintain consistent tumor growth, only mice between 4 and 6 weeks ofage were used.

2. Tumor Implantation And Measurement

Transformed cells obtained by oncogene transfection of NRKE cells asdescribed in Example 47 above were grown as xenograft tumors in thefemale CD1 nu/nu mice. Transformed cells grown in vitro through passage7 were used to establish the xenografts. The Lewis lung carcinoma wasgrown as a syngeneic tumor in C57B1/6 mice as described previously inMerriman et al., Cancer Research, 49, 4509-4516 (1989). Tumors wereinitiated by the subcutaneous implantation of 1×10⁶ cells approximately1 cm from the first mammary gland. After implantation, the mice wererandomized and divided into treatment groups of 10 mice per group.Starting one day after tumor implantation, the mice were dosed IP withdrug daily for 8 days as described in the next section. On day 10, tumormass was determined as described by Tomayko and Reynolds, CancerChemother. Pharmacol., 24, 148-154 (1989) with the following ellipsoidvolume equation:Mg tumor=½ (length×width×height)

Percent inhibition of tumor growth was calculated from the ratio of thetumor mass for the drug treated animals relative to the mass for thevehicle treated animals. All animals were weighed at the beginning andend of treatment to determine if inhibition of tumor growth was due toweight loss.

Adenocarcinomas are produced in female CDl nu/nu nude mice approximately3 days after the subcutaneous implantation of 1×10⁶ cells. These tumorsgrow with a doubling time of 24 hours. In contrast, no tumors areproduced in nude mice up to one year after the subcutaneous implantationof 108 parent NRKE cells.

3. Drug Treatment

CY was dissolved in isotonic saline, 20 and 21 were dissolved in cornoil, and 1 was dissolved in 10 percent emulphor 620 in Dulbecco'sphosphate buffered saline. It should be noted that 1 was poorly solublein this vehicle. Starting 1 day after tumor implantation, drugs wereadministered interperitoneally (IP) at the doses given in Table X. CYand compounds 20 and 21 were given once daily for 8 days. Compound 1 wasadministered in three doses per day at lower dose levels, and theanimals were treated for 7 days. On day 10, tumor masses, percentinhibition of tumor growth, and weight changes were determined.

4. Results

The results, presented in Table X, show that the three lavendamycinanalogs 1, 20 and 21 gave excellent tumor growth inhibition in vivo.Other investigators have found that a dose of 100 mg/kg/day oflavendamycin and related compounds is lethal, but compounds 1, 20 and 21showed little or no toxicity even at doses up to 300 mg/kg/day. Theweight loss induced by compound 20 may be due to the presence of theisoamyl group, and replacement of this group by groups having an evennumber of carbons may reduce the slight toxicity. Since compounds 1 and21 showed no toxicity, use of higher dose levels may increase theinhibition of tumor growth with little or no toxicity.

TABLE X Number Dose Dead (Mg/Kg/ {overscore (Number)} Percent Wt.Percent Inhibition Compound Day) Treated Change (g) Of Tumor Growth 20150 0/7 −11.5 ″ 0.05  88 ″ 5  300 0/7 −15.7 ″ 0.08  89 ″ 12 21 150 0/7+2.0 ″ 0.12 34 ″ 16 300 0/7 −1.0 ″ 0.05 78 ″ 13 CY 60 0/7 −3.0 ″ 0.09 97″ 2   1 10 0/7 +8.4 ″ 0.68 10 ″ 14 30 0/7  +9 ″ 0.54 26 ″ 18 100 0/7+2.5 ″ 1.70 69 ″ 5 

Example 49 National Cancer Institute Testing

The following compounds were submitted for testing in the NationalCancer Institutes (NCI's) cancer drug discovery and development program:1, 2, 16, 20, 21, 26, 30, 55, 7-N-formyldemethyllavendamycin octyl ester(56), demethyllavendamycin ester (57), and 6-Chlorolavendamycin methylester (58). The preparation and structures of compounds 1, 2, 16, 20,21, 26 and 30 are provided in examples 1, 2, 16, 20, 21, 26 and 30. Thepreparation and structure of compound 55 is provided in Example 47. Thepreparation and structure of compound 58 is provided in Example 52.

Compound 56 and the intermediate compounds in its preparation wereprepared as described above. In the final step, 34.2 mg (0.148 mmole) of2-formyl-7-formamyl quinoline-5,8-dione and 48.02 mg (0.152 mmole) oftryptophan octyl ester were mixed in a three-neck round bottom flaskequipped with reflux apparatus and a magnetic stirrer. The flask washeated in an oil bath; the oil bath temperature was raised from roomtemperature to 127° C. over three hours and then was maintained at 127°C. for 32 hours. The reaction was stopped after 32 hours, and thesolution was hot filtered. A brown-yellow solid was obtained. H¹NMR wasperformed with the result of *0.88 (t, 3H), *1.3 (m, 10H), *1.9 (m, 2H),*4.8 (t, 2H), *7.66 (t, 1H), 7.70 (t, 1H), *7.74 (d, 1H), 7.98 (s, 1H),*8.24 (d, 1H), *8.53 (s, 1H), *8.55 (s, 1H), *8.75 (s, 1H), *8.95 (s,1H), *9.21 (s, 1H), *11.75 (s, 1H).

Compound 57 is identical to 56, except that it has —NH₂ at position 7instead of —NHCOH. It was prepared by acid hydrolysis of 56.

The NCI's cancer drug discovery and development program is described inGrever et al., Seminars in Oncology, 19, 622-38 (1992). Compounds chosenfor the program are initially tested in an in vitro anticancer screen. Atotal of fifty-five human tumor cell lines, derived from nine cancertypes (lung, colon, melanoma, renal, ovarian, brain, prostate, breastand leukemia), are used in this screen. The tumor cells are inoculatedover a series of standard 96-well microtiter plates on day 0. Thesecells are then preincubated on the microtiter plates for 24 hours. Thetest compounds are added to the wells in five 10-fold dilutions startingfrom the highest soluble concentration. Each compound is tested againstevery cell line in the panel. The compounds are incubated for 48 hourswith the tumor cell lines. At the termination of the assay, the cellsare fixed in situ, washed, and dried. Sulforhodamine B (SRB), a pinkanionic dye that binds to the basic amino acids of trichloroaceticacid-fixed cells, is added. The cells are washed again, and theremaining dye is a function of the adherent cell mass. The bound stainis solubilized and measured spectrophotometrically. The data are enteredfrom automatic reading devices into a computer for subsequent storageand analysis.

The measured effect of each test compound on the cell lines iscalculated according to one or the other of the following twoexpressions:

-   If (Mean OD_(test)−Mean OD_(tzero))□ 0, then PG=100×(Mean    OD_(test)−Mean OD_(tzero))/(Mean OD_(ctrl)−Mean OD_(tzero)); or-   If (Mean OD_(test)−Mean OD_(tzero))<0, then PG=100×(Mean    OD_(test)−Mean OD_(tzero))/Mean_(tzero);    Where:-   PG=percent growth.-   Mean OD_(tzero)=The average of optical density measurements of    SRB-derived color just before exposure of cells to the test    compound.-   Mean OD_(test)=The average of optical density measurements of    SRB-derived color after 48 hours exposure of cells to the test    compound.-   Mean OD_(ctrl)=The average of optical density measurements of    SRB-derived color after 48 hours with no exposure of cells to the    test compound.

The NCI provides the following information about the results of thecancer screen for each test compound. First, a data sheet is providedwhich lists, for each cell line, the Mean OD_(tzero), the MeanOD_(ctrl), the Mean OD_(test) for each of the five concentrations, thecalculated PGs for each concentration, and the response parameters G150,TGI, and LC50. G150, TGI, and LC50 are interpolated values representingthe concentrations at which the PG is +50, 0, and −50, respectively.

Second, dose-response curves are provided. These curves are created byplotting the PGs against the log₁₀ of the corresponding concentrationfor each cell line. Horizontal lines are provided at the PG values of+50, 0, and −50. The concentrations corresponding to points where thecurves cross these lines are the G150, TGI, and LC50, respectively.

Third, mean graphs at each of the principal response parameters (G150,TGI, and LC50) are provided. Bars extending to the right of a verticalline representing the mean for each of these parameters showssensitivity of the cell line to the test agent in excess of the averagesensitivity of all tested cell lines. Since the bar scale islogarithmic, a bar two units to the right implies the compound achievedthe response parameter (eq., G150) for the cell line at a concentration{fraction (1/100)} the mean concentration required over all cell lines,and thus the cell line is unusually sensitive to that compound. Barsextending to the left correspondingly imply sensitivity less than themean. Thus, the mean graphs facilitate visual scanning of data forpotential patterns of selectivity for particular cell lines or forparticular subpanels with respect to a selected response parameter.

Finally a dose-response matrix is provided for each test compound. Itcombines some qualities of the dose-response curve with some qualitiesof the mean graph. Selective effects at the cell line or subpanel levelsare visualized as in the mean graph, however, different levels of effectare also depicted simultaneously, as in dose response curves.

The values of DG₁₅₀, DTGI, and LC50 displayed near the bottom of thedose-response matrix are measures of subpanel selectivity based on theresponse parameters G150, TGI, and LC50. These values identify whethersubpanel-selective effects occur at a high (LC50 or TGI) or moderate(G150) effect level. Computer simulations suggest that a value ofD_(G150), D_(TGI) or D_(LC50)□ 50 is statistically significant. Tocalculate D_(G150), the percentage of cell lines that achieve G150 foreach subpanel is calculated separately for each concentration. Thenthree differences are calculated separately for each of the 5concentrations: (1) the highest subpanel percentage minus the percentageof the remaining cell lines, (2) the percentage for the 2 highestsubpanels, taken together, minus the percentage for the remaining celllines, and (3) the average of the 3 highest subpanel percentages minusthe percentage for the remaining cell lines, D_(G150) is the largest ofthese 15 differences. The values D_(TGI) and D_(LC50) are the analogousmaximum differences relating to TGI and LC50.

The value of DH at the bottom of the dose-response matrix provides amore general measure of selective effect. The DH value is givenprimarily as a means of assigning relative scores of selectivity to thecompounds, and the practical significance of this value will bedetermined empirically. However, computer simulations suggest thatvalues of D_(H)□75 are statistically significant. To calculate D_(H),each cell line is ranked 1 to N (where N is the total number of cellline tests which satisfy quality control criteria) from least to mostsensitive, by PG value. This is done separately for each concentration.At each concentration, a mean rank is calculated for each subpanel byaveraging the individual cell line ranks, and the subpanels are orderedby sensitivity as measured by mean rank. At each of the 5concentrations, the 3 mean rank differences are calculated: the meanrank for the k (where k=1, 2, 3) most sensitive subpanels, takentogether, minus the mean rank of the remaining cell lines. D_(H) is thelargest of these 15 differences, multiplied by 200/N, so that its rangeis 0-100.

The maximum of D_(G150), D_(TGI) and D_(LC50) is used to determinewhether subpanel-selective cytotoxicity is occurring most markedly atthe G150, the TGI, or the LC50 level, and thus to determine which of thethree corresponding mean graphs to display on the dose-response matrix.The MGD_(H) value is a measure of subpanel selectivity similar to DH,but relating to this chosen mean graph. Each cell line is ranked, fromleast to most sensitive, by the mean graph bar extension. MGD_(H) is themaximum difference in mean rank between the most and least sensitivesubpanels, which are maximized over the choice of 1, 2, or 3 subpanelsto be the most sensitive, and multiply by 200/N, just as in thecalculation of D_(H). The MGD_(H) value is given primarily as a means ofassigning relative scores to the mean graphs of the compounds. However,computer simulations indicate that values exceeding 75 are statisticallysignificant.

Numerous patterns of cellular responsiveness are generated by the manysubstances that are tested in the NCI cancer screen. Grever et al.,Seminars in Oncology, 19, 622-38 (1992) reports that application ofcomputerized programs and neural network analysis of the primary invitro screening data have provided evidence that it is identifyingcompounds with some biological relevance.

From the above discussion, it can be seen that the data provided by theNCI for each compound is substantial and complicated. Consequently, noattempt will be made here to present all of the data for the tencompounds submitted to the NCI for testing. Instead, Table XI belowprovides a summary of the activity of each of the ten compounds againstthe nine types of cancer. Also, Table XII lists those compounds whichshowed selectivity toward particular types of cancer. In particular,compounds 16 and 55 exhibited unexpected selectivity for renal cancerand non-small cell lung cancer, respectively. The complete package ofdata received from the NCI and more information about the calculationand interpretation of the data will be filed as part of an informationdisclosure statement during the prosecution of this application.

TABLE XI Activity (Average PG for highest dose tested)* Non-Small CellColon CNS Ovarian Renal Prostate Breast Compound Leukemia Lung CancerCancer Cancer Melanoma Cancer Cancer Cancer Cancer 1 +36.3 +42.6 +80.0+60.2 +80.3 +70.0 +63.0 +89.5 +47.9 2 −34.7 −27.9 −43.6 −26.6 −53.7−62.7 −74.6 −45.5 −32.6 16 −28.3 −52.3 −52.1 −60.4 −78.4 −55.5 −72.9−18.0 −45.1 20 −4.0 −19.1 −53.0 −49.5 −75.2 −42.2 −56.1 −70.5 −38.6 21+20.3 +13.0 +62.3 +59.3 +58.6 +47.0 +67.5 +69.5 +62.9 26 −10.8 −50.9−17.9 −61.0 −79.0 −63.2 −93.0 −81.5 −35.0 30 −21.7 −71.1 −66.6 −93.8−85.1 −86.7 −92.0 −79.0 −62.7 55 +30.0 −60.8 −20.0 −39.3 −53.0 −43.5−63.8 −31.0 −43.8 56 −29.7 −28.8 −30.0 −36.8 −39.9 −57.3 −51.3 +14.0−42.3 57 +90.8 +89.8 +100.3 +81.0 +89.4 +89.3 +68.7 +94.5 +96.0 58 −14.6−69.3 −84.9 −52.2 −86.1 −39.3 −73.7 −63.0 −58.7 *See above forcalculation of PG. The average PG was calculated by summing the PGs forindividual cell lines and dividing the sum by the number of cell lines.A PG below +100% indicates activity.

TABLE XII Compound Selectivity 16 One of the seven renal cancer celllines was about 100 times more sensitive to compound 16 than the averagesensitivity of the fifty-five cell lines at one or more dose levels(i.e., at GI50, TGI and/or LC50).* 55 One of the seven non-small celllung cancer cell lines was about 100 times more sensitive to compound 55than the average sensitivity of the fifty-five cell lines at one or moredose levels (i.e., at GI50, TGI and/or LC50).* *A bar two units to theright of the mean line on the mean graphs indicates this level ofsensitivity (see above).

As part of the NCI's cancer drug discovery and development program,certain compounds shown to have antitumor activity in the cancer screenare referred to the Biological Evaluation Committee for Cancer Drugs(BEC/Cancer) for secondary in vitro and in vivo investigations that arecritically important to determining whether they have the potential forbecoming actual anticancer drugs. The BEC/Cancer reviews the in vitroantitumor data, determines the further secondary testing that isrequired, and initiates the in vivo evaluations to assess thetherapeutic index (i.e., an assessment of the antitumor effects inconjunction with the observed toxic effects).

As a result of its performance in an initial and a repeat cancer screen,compound 55 has been selected for in vivo testing. Compound 55 waschosen primarily because of its selectivity for non-small cell lungcancer. To date, toxicity studies have been performed, and the maximumtolerated dose in mice has been determined to be 400 mg/kg.

Also, compounds 2, 16, 26 and 30, which were submitted to the NCI muchlater than compound 55, were selected for repeat testing in the cancerscreen. As a result of this repeat cancer screen, compounds 16, 26 and30 are being considered by the BEC for in vivo testing. It should benoted that in the repeat screen compound 16 did not exhibit theselectivity for renal cancer seen in the initial screen and compound 30showed selectivity for leukemia.

Compound 55 does not come within the scope of the formulas of theclaimed compounds of the invention. However, it may be utilized to treatcancer in the same manner described above for the claimed compounds,i.e., it may be made into suitable pharmaceutical compositions, may beadministered, and effective dosages may be determined, all as describedabove for the claimed compounds.

Example 50 Activity Against Leishmania

Leishmania are parasitic protozoa (family Trypanosomatidae) which arethe causal agents of leishmaniasis in mammals. Three7-N-acetyllavendamycin esters were tested for activity againstLeishmania major. The three compounds tested were 1, 20 and 21 (forpreparation and structure of these compounds, see Examples 1, 20 and21).

An in vitro assay was performed as follows. L. major promastigotes wereincubated at 1×10⁶ promastigotes per ml in 5 ml of culture medium(medium 199 containing 20% heat-inactivated fetal bovine serum) forthree days at 26EC in the presence of 0.5 to 10.0 FM of compound 1, 20or 21 or of pentamidine isethionate (1,5-bis[p-amidinophenoxyl]-pentanebis[2-hydroxyethane sulfonate]; Sigma, product number PO₅₄₇).Pentamidine isethionate is a widely used anti-leishmanial therapeuticdrug. The drugs were initially solubilized in alcohol, DMSO or salineand then diluted in fresh culture medium to the desired concentration.Control cultures received diluent only.

After the three days of culture, viable parasites were counted on ahemocytometer using a light microscope. Multiple counts of duplicatewells were made. The results are presented in FIG. 1A. The percentsurvival was calculated as:$\frac{{Number}\quad{of}\quad{cells}\quad{in}\quad{culture}\quad{with}\quad{drug}}{{Number}\quad{of}\quad{cells}\quad{in}\quad{culture}\quad{without}\quad{drug}} \times 100$

The cells were then washed by centrifuging them at 1200 rpm. Freshculture medium was added, and the cells were incubated for an additionalthree days as described above. Viable promastigotes were counted asbefore, and the results are presented in FIG. 1B.

Compound 1 dissolved in corn oil was administered intraperitoneally toBalb/c mice (Ball State University colony) at 80 mg/kg/day for sevendays beginning one day prior to injection of 5×10⁶ L. majorpromastigotes in medium 199 in the hind foot. Control mice received anequivalent amount of corn oil and L. major. Beginning on day 8, thethickness of the footpads top to bottom was measured using a verniercaliper Footpad swelling indicates progression of the disease. Thepercent increase in footpad size was calculated as:$\frac{{{Infected}\quad{Footpad}\quad{Size}} - {{Normal}\quad{Footpad}\quad{Size}}}{{Normal}\quad{Footpad}\quad{Size}} \times 100$The results are presented in FIG. 2. The percent increase in footpadsize is for triplicate mice in the control group and duplicate mice inthe treated group. Finally, the mice did not appear to be adverselyaffected by the administration of compound 1 as measured by weight loss.

Example 51 In Vitro Cytotoxicity

Lavendamycin analogs 2-16 and 58-61, quinolinediones 23-30 andlavendamycin methyl ester (55) were screened for antitumor activity asdescribed in Example. 47. The preparation and structures of lavendamycinanalogs 2-16 are set forth in Examples 2-16. The preparation andstructures of quinolinediones 23-30 are set forth in Examples 23-30. Thepreparation of lavendamycin methyl ester (55) is described in Example47. Lavendamycin analogs 58-61 were prepared from the correspondingaldehydes and tryptophans as described above. For experimentalconditions and yields, see Table XIII below. For analytical data, seeTable XIV below. The results of the screening are presented in Tables XVand XVI below.

TABLE XIII Compd. R¹⁰ Y R¹ R⁶ Solvent Hours (° C.) Yield (%) 58 (CH₂)₇CHCO₂CH₃ H H Xylene 3 (25-130°) 62.7 4 (130°) 59 CH₃ CH₂OH H H Anisole 3(room-160N) 48 60 CH₃ CO₂CH₃C₆H₅ H H Xylene 3 (25-130°) 58 16.5(130-135°) 61 CH₃ CO₂CH₂CH₃ H H Xylene 6 (25-167N) 54.8 32 (107°)

TABLE XIV Compound R¹⁰ Y R⁴ R⁶ ¹H, NMR MS HRMS 58 (CH₃)₂CH CO₂CH₃ H H1.4(d, 6H), 2.75(xt, 1H), 4.09(s, 3H), 7.41(dd, 468 (40), 452 (100), 394(45) C₂₆H₂₀N₇O₅ 1H), 7.81-7.67(m, 2H), 7.82(s, 1H), 8.0(s, 1S), Calc468.14471 8.38(dd, 1H), 8.50(d, 1H), 8.97(s, 1H), 9.06(d, Found468.14229 1H), 11.37(s br, 1H) 59 CH₃ CH₂OH H H (DMSO) 2.30(s, 3H),375-4.1(AB, 2H), 7.13 415 (M+3⁺, 60), 414 (M+2⁺, (d, 1H), 7.31(t, 1H),7.45(d, 1H), 7.68(d, 1H), 55), 413 (M+1⁺, 70), 412 7.81(s, 1H), 8.49(d,1H), 8.61(d, 1H), 10.3(s, (M⁺, 100) 1H), 11.2(s, 1H) 60 CH₃ CO₂CH₂C₆H₅ HH 2.35(s, 3H), 5.5(s, 2H), 7.26(s, 5H), 7.43(t, 1H), 7.61(d, 1H),7.70(t, 1H), 7.97(s, 1H), 8.22(s, 1H), 8.40(s br, 1H), 8.51(d, 1H),8.96(s, 1H), 9.17(d, 1H). 61 CH₃ CO₂CH₂CH₃ H H 1.52(t, 3H), 2.31(s, 3H),4.53(q, 2H), 7.34(d, C₂₅H₁₆N₄O₅ 1H), 7.64-7.61(2H), 7.91(s, 1H), 8.18(d,1H), Calc 454.1277 8.34(s, 1H), 8.46(d, H), 8.89(s, 1H), 9.10(d, Found454.1255 1H), 11.69(s br, 1H)

TABLE XV LC50 (:M) Compound NRKE K/1 H/1.2 N/4.2 3LL 2 0.15 0.07 0.110.10 0.40 3 0.70 0.40 0.69 0.70 1.0 4 2.5 2.2 2.1 2.2 >3.3 5 0.62 0.330.70 0.84 2.1 6 0.40 0.44 0.40 0.49 3.0 7 0.58 0.79 0.58 0.78 2.0 8 0.960.52 0.74 1.05 2.3 9 1.35 0.56 1.32 1.2 3.2 10 >33.015.0 >33.0 >33.0 >33.0 11 0.32 0.33 0.40 0.42 0.82 12 0.66 0.50 0.410.70 2.3 13 1.0 0.6 0.37 0.65 2.4 14 >33.0 3.3 16.0 15.0 16.0 15 0.900.40 0.61 0.50 0.9 16 0.90 0.35 0.23 0.22 0.21 23 5.0 12.5 5.5 7.0 3.724 3.6 5.8 3.4 3.2 2.1 25 >3.3 2.15 >3.3 >3.3 1.0 26 3.0 >3.3 3.3 >3.32.5 27 4.6 14.0 5.0 5.7 2.6 28 3.2 6.6 2.8 3.4 2.3 29 1.5 7.5 1.45 1.150.71 30 2.0 >3.3 2.7 >3.3 2.2 55 0.1 0.03 0.11 0.06 0.25 56 >3.32.1 >3.3 2.2 3.2 58 0.70 0.50 0.66 0.64 1.3 59 0.25 0.20 0.20 0.22 0.4060 9.50 9.50 >10.0 >33.0 >33.0 61 0.30 0.04 0.38 0.20 3.2

TABLE XVI Differential Index Compound K/1 H/1.2 N/4.2 3LL 2 2.14 1.361.50 0.38 3 1.75 1.01 1.00 0.70 4 1.14 1.19 1.14 <0.75 5 1.88 0.88 0.740.29 6 0.91 1.00 0.82 0.13 7 0.73 1.00 0.76 0.29 8 1.85 1.30 0.91 0.42 92.41 1.02 1.13 0.42 10 >2.2 11.0 11.0 11.0 11 0.97 0.80 0.76 0.39 121.32 1.61 0.94 0.29 13 1.67 2.70 1.53 0.42 14 >10.0 >2.06 >2.2 >2.06 152.25 1.48 1.80 1.00 16 2.57 3.91 4.09 4.29 23 0.4 0.9 0.7 1.35 24 0.61.06 1.125 1.7 25 >1.5 −1.0 −1.0 >3.3 26 <0.9 0.9 <0.9 1.2 27 0.3 0.90.8 1.8 28 0.48 1.1 0.9 1.4 29 0.2 1.03 1.3 2.1 30 <0.6 0.7 <0.6 0.9 553.33 0.9 1.67 0.4 56 >1.57 11.00 >1.5 >1.03 58 1.40 1.07 1.09 0.54 591.25 1.25 1.14 0.63 60 1.00 <0.95 <0.29 <0.29 61 7.5 0.83 1.5 0.09 NRKE= Normal Rat Kidney Epithelial Cells. K/1 = ras ^(K) Transformed NRKECells. H/1.2 = ras ^(H) Transformed NRKE Cells. N/4.2 = ras ^(N)transformed NRK cells 3LL = Lewis Lung Carcinoma Cells

Example 52 Synthesis of 6-Chlorolavendamycin Methyl Ester (58)

7-N-Acetyllavendamucin methyl ester (AA) in 60 ml dried methanol wasstirred and heated in a 60° C.-65° C. oil bath. While a stream of dryhydrogenchloride was being passed through the solution for 21 hrs. (SeeEquation 10 below) The reaction mixture was immediately rotaryevaporatedto dryness to give 64.3 mg (88%) of an orange solid. Mp: 270° C. (dec).¹H NMR (CDCl₃) δ 11.88(s, 1H), 9.09(d, 1H), 8.60(d, 1H). 8.37(d, 1H),7.79(d, 1H), 7.77(s, 1H), 7.65(t, 1H), 7.40(t, 1H), 4.07(s, 3H), 3.21(s,3H).

This compound 58 was tested in the NCI In Vitro Screening described inExample 49 above and also in the NCI Hollow Fiber Assay For PreliminaryIn Vivo Testing described in Example 55 below.

Example 53 Synthesis of 7-Amino-6-Chloro-2-Methylquinoline-5.8-Dione(59)

A slow stream of hydrogen chloride gas was passed through a solution of7-acetamido-2-methylquinoline-5,8-dione (6, 230 mg, 1 mmol) in drymethanol (15 ml) at 60° C. for 48 h. (See Equation 11 below) Theresulting acidic solution was neutralized with 2% sodium bicarbonate (10ml), extracted with CH₂Cl₂ (5×30 ml) and dried over sodium sulfate (2.0g, 20 min). Evaporation of the solvent in vacuo afforded 98 mg (42%) ofpure (59) as red crystals: mp 270° C. (Dec., MeOH); ¹H NMR (CDCl3) δ8.31 (1H, d, J=8.0 Hz, H-4), 7.47 (1H, d, J=8.0 Hz, H-3), 5.70 (2H, brs,NH₂), 2.68 (3H, s, CH₃); IR (KBr) v_(max) 3471, 3329, 3191, 3085, 2960,2924, 1649, 1605, 1587, 1573, 1382, 1370, 1326, 1273, 1259, 1164, 737cm⁻¹; EIMS, m/e (relative intensity) 223 (M+1, 100), 189 (4.1), 133(0.4), 81 (0.8), 69 (1.2); EIMS, m/e (relative intensity) 224 (M+2,27.3), 222 (M+, 97.2), 195 (7.4), 187 (M-CI, 100), 166 (5.3), 131(10.4), 104 (12.1), 77 (14.9), 64 (10.5); HRMS, m/e for C₁₀H₇ClN₂O₂calcd 222.0196, found 222.0194.

Example 54 Synthesis of 6-Chloro-7-Amino-2-Formylquinoline-5.8-Dione(60)

A stirred mixture of 6-Chloro-7-amino-2-methylquinoline (2) (267.6 mg,1.2 mmol) and SeO₂ (213 mg, 1.92 mmol) in dry dioxane (40 ml) and water(0.18 ml) under Ar was heated at reflux for 23 hrs. (See Equation 12below) The mixture was not filtered and the residue on the filter paperwas washed with 40 ml chloroform. Evaporation of the filtrate gave a redresidue. This residue was dissolved in 480 ml of chloroform, washed with4×60 ml sodium chloride saturated solution and dried with anhydrousMgSO₄. After evaporation it gave a red solid (3) 209 mg (74%). Mp: 211°C. (dec), ¹H NMR (CDCl₃) δ 10.24(s, 1H), 8.63(d, 1H), 8.25(d, 1H),5.6-6.0(broad, NH).

Example 55 Synthesis of Demethyllavendamycin Octyl Ester

The following compound (61) was synthesized by acid hydrolysis ofcompound 21 and then characterized by spectroscopic methods.

Example 56 NCI Hollow Fiber Assay for Preliminary in Vivo Testing

The biological Testing Branch of the Developmental Therapeutics Programof the National Cancer Institute has adopted a preliminary in vivoscreening tool for assessing the potential anticancer activity ofcompounds identified by the large scale in vitro cell screen. For theseassays, human tumor cells are cultivated in polyvinylidene fluoride(PVDF) hollow fibers, and a sample of each cell line is implanted intoeach of two physiologic compartments (intraperitoneal and subcutaneous)in mice. Each test mouse receives a total of 6 fibers (3intraperitoneally and 3 subcutaneously) representing 3 distinct cancercells lines. Three mice are treated with potential antitumor compoundsat each of 2 test doses by the intraperitoneal route using a QD×4treatment schedule. Vehicle controls consist of 6 mice receiving thecompound diluent only. The fiber cultures are collected on the dayfollowing the last day of treatment. To assess anticancer effects,viable cell mass is determined for each of the cell lines using aformazan dye (MTT) conversion assay. From this, the % T/C can becalculated using the average optical density of the compound treatedsamples divided by the average optical density of the vehicle controls.In addition, the net increase in cell mass can be determined for eachsample as a sample of fiber cultures are assessed for viable cell masson the day of implantation into mice. Thus, the cytostatic and cytocidalcapacities of the test compound can be assessed.

Generally, each compound is tested against a minimum of 12 human cancercell lines. This represents a total of 4 experiments since eachexperiment contains 3 cell lines. The data are reported as % T/C foreach of the 2 compound doses against each of the cell lines withseparate values calculated for the intraperitoneal and subcutaneoussamples.

Compounds are selected for further in vivo testing in standardsubcutaneous xenograft models on the basis of several hollow fiber assaycriteria. These include: (1) a % T/C of 50 or less in 10 of the 48possible test combinations (12 cell lines X2 sites X2 compound doses);(2) activity at a distance (intraperitoneal drug/subcutaneous culture)in a minimum of 4 of the 24 possible combinations; and/or (3) a net cellkill of 1 or more cell lines in either implant site. To simplifyevaluation, a points system has been adopted which allows rapid viewingof the activity of a given compound. For this, a value of 2 is assignedfor each compound dose which results in a 50% or greater reduction inviable cell mass. The intraperitoneal and subcutaneous samples arescored separately so that criteria (1) and (2) can be evaluated.Compounds which meet the guidelines of the Biological evaluationCommittee for Cancer Drugs (BEC/C) for Further Testing, namely acombined IP+SC score >20, a SC score >8 or a net cell kill of one ormore cell lines, are referred for evaluations in subcutaneous humantumor xenograft assays. These criteria were statistically validated bycomparing the activity outcomes of >80 randomly selected compounds inthe hollow fiber assay and in the xenograft testing. This comparisonindicated that there was a very low probability of missing an activecompound if the hollow fiber assay were used as the initial in vivoscreening tool. In addition to these criteria, other factors (e.g.unique structure, mechanism of action) may result in referral of acompound for standard xenograft testing without the compound meetingthese criteria.

Compound 58 (6-chlorolavendamycin methyl ester) was submitted to thistest and received the following scores:

-   -   IP Score: 10    -   SC Score: 4    -   Total Score: 14    -   Cell Kill: N

Example 57 Anti-HIV Reverse Transcriptase Activity

Compound Nos. 1, 2, 12, 16, 17, 20, 21 and 61 were tested for anti-HIVreverse transcriptase activity. In addition, lavendamycin methyl ester(Compound B above) and streptonigrin (Compound G above) were likewisetested. The inhibition of HIV-RT activity was measured by a modificationof the methods of Hoffman et al (1985), Take et al (1989) and Robbins etal (1994). Hoffman, A.; Bananpour, B.; Levy, J. Characterization of theAIDS-associated retrovirus reverse transcriptase and optimal conditionsfor its detection in virions. Virology, 1985, 147, 326. Take, Y.;Inouke, Y.; Nakamura, S.; Comparative studies of the inhibitoryproperties of Antibiotics on human immunodeficiency virus and cellularDNA polymerases. Journal of Antibiotics 1989, 42, 107. Robbins, B.;Rodman, J.; McDonald, C.; Srinivas, R.; Flynn, P.; Fridland, A.;Enzymatic Assay for the measurement of Zidovudine Triphosphate inperipheral blood mononuclear cells. Antimicrobial Agents and Actions1994, 38, 115.

The procedure was as follows: The reaction mixture (100 μl) consisted of100 mM Tris-HCL(pH 8.0), 5 mM dithiothreiotol, 5 mM MgCl₂, 60 mM NaCl, 4μg/ml poly(rA)-oligo(dt)₁₂₋₁₈, 0.2 mM[³H]TTP and 1.0 units/ml of HIV-RT(Worthington Biochemical).

The samples to be tested were initially dissolved in DMSO at no morethan 1 mg/ml and then serially diluted with distilled water to providedifferent concentrations of test solutions. A mixture of assay solutioncontaining all the above reactants, buffers and salts in 8×concentration was added to each test solution to give the final reactionmixture in a total of 300 μl. For each test solution this reactionmixture was divided into three aliquots of 100 μl each and incubated ona 96 well flat-bottom microtiter plate at 37° C. for 1 hour. Thereaction was terminated with 25 μl of 0.1M EDTA on ice and 30 μl fromeach test well was spotted on 1 cm square of DE 81 ion exchange paper(Whatman). Each filter paper square was then washed 3 times with 5%TCA-NaHPO₄. 12H₂O and then one time each with dd H₂O and 95% ethanol.The squares were washed sequentially in beakers for 18-20 minutes withswirling. Streptonigrin (3 μg/ml) or AZT-TriPO₄ (1 μg/ml) was used aspositive inhibitory controls. As a negative control, no enzyme was addedto the reaction mixture. The squares were then dried and added to vialswith scintillation fluid and the amount of radioactive uptake measuredon a Beckman liquid scintillation counter.

The inhibitory capacity of each analog at each of the three enzymeconcentrations mentioned above was determined at 4 different drugconcentrations ranging from 15 to 0.125 μg/ml by comparing the meanenzymatic incorporation of [³H]TTP in the presence of each drug to themean incorporation in the absence of the drug. The concentration percentof the enzymatic activity is inhibited (IC₅₀) was determined by simplelinear interpolation from this data for each enzyme concentration. TheseIC₅₀ determinations were repeated three times at least and evaluated forvalidity against our positive standards. The results are shown in TableXVII below;

TABLE XVII IC₅₀ for Compound HIV-RT (mM) 21 10.62 12 12.28 20 18.54  222.39  1 26.84 16 27 17 >30 61 18.19 B >30 Streptonigrin 21.35

In order to determine any possible additive or synergistic inhibitoryactivity which the lavendamycin analogs might have when combined withAZT, a range of concentrations of both 3 azido 3′-deoxythymidine5′-triphosphate (AZT-TP) and a lavendamycin analog at levels equal orbelow the IC₅₀ concentration for either drug alone were tested asdescribed above. AZT-TP was used for these in vitro studies since AZT isnaturally phosphorylated in vivo.

The results of these tests are given in Table XVIII and XIX below, wherethe percent inhibition of HIV-RT activity is reported for threedifferent trials and then averaged in the seventh column. For thoseexperiments which used a combination of AZT-TP and a lavendamycinanalog, the eighth column shows the arrearage percent inhibitionmeasured for the combination minus the average percent inhibition forthe same concentration of each of the compounds singly. Thus, a positivenumber in this column shows a synergistic effect, namely the combinationproduced a higher percent inhibition than the sum of the compoundsalone. Likewise, a negative number shows an antagonistic effect.

TABLE XVIII LA Conc. AZT-TP % inh. % inh. % inh. % inh. Compared Drug(μM) Conc. (μM) Trial 1 Trial 2 Trial 3 ave. to Add. AZT-TP 0 0.05 51 5249 50.7 AZT-TP 0 0.01 27 27 29 27.7 16 6.0 0 26 33 30 29.7 16 3.0 0 0 312 5.0 16 .5 0 0 0 0 0.0 AZT-TP + 16 6.0 0.05 70 67 69 68.7 −11.7AZT-TP + 16 3.0 0.05 70 67 69 68.7 13.0 AZT-TP + 16 0.5 0.05 53 62 5857.7 7.0 AZT-TP + 16 6.0 0.01 55 51 50 52.0 −5.3 AZT-TP + 16 3.0 0.01 4747 30 41.3 8.7 AZT-TP + 16 .5 0.01 48 40 42 43.3 15.7

TABLE XIX LA Conc. AZT-TP % inh. % inh. % inh. % inh. Compared Drug (μM)Conc. (μM) Trial 1 Trial 2 Trial 3 ave. to Add. AZT-TP 0 .05 56 48 5051.3 AZT-TP 0 0.01 18 17 14 16.3 20 0.5 0 5 6 20 10.3 21 0.5 0 13 6 2815.7  2 0.5 0 12 5 17 11.3 AZT-TP + 20 0.5 0.05 63 59 66 62.7 1.0AZT-TP + 21 0.5 0.05 64 69 63 65.3 −1.7 AZT-TP + 2  0.5 0.05 69 69 7069.3 6.7 AZT-TP + 20 0.5 0.01 41 42 50 44.3 17.7 AZT-TP + 21 0.5 0.01 5055 44 49.7 17.7 AZT-TP + 2  0.5 0.01 59 47 46 50.7 23.0

As seen above, some combinations of the lavendamycin analogs with AZT-TPresulted in RT inhibition which reflected simply an additive effect ofthe two drugs acting independently to inhibit the enzymatic activity.These combinations resulted in a total combined activity which wasapproximately +/−5% of the expected additive effect.

A few of the combinations resulted in synergistic activity where thecombined activity significantly exceeded the additive effect of eitherdrug alone, namely >5% of the expected additive effect.,

In general the best combined activity with lavendamycin analogs wasobserved at concentrations of AZT-TP lower than the IC₅₀ dose. At higherlevels of AZT-TP the drug combinations appeared to interactantagonistically giving less than an additive effect.

1. A compound having the following formula:

wherein,

NH₂, Y is H, OR¹¹, SR¹¹, N(R¹¹)₂, NR¹¹N(R¹¹)₂, a halogen atom, NO₂, CN,

an alkyl, aryl, cycloalkyl, alkynyl, alkenyl or heterocyclic residue,each of which may be substituted or unsubstituted, R¹ is a halogen atom,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, which may be the same or different, eachis independently H, a halogen atom, NO₂, CN, OR¹³, SR¹³, N(R¹³)₂,

an alkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclic,heteroalkenyl or heteroalkynyl residue, each of which may be substitutedor unsubstituted, R⁹ is H,

an alkyl, cycloalkyl, aryl, alkenyl, alkynyl or heterocyclic residue,each of which may be substituted or unsubstituted, R¹⁰, R¹¹ and R¹³,which may be the same or different, each is independently H or an alkyl,cycloalkyl, alkenyl, alkynyl, aryl or heterocyclic residue, each ofwhich may be substituted or unsubstituted, R¹² is H, N(R¹¹)₂, OR¹¹,SR¹¹, NR¹¹N(R¹¹)₂, OR¹⁴N(R¹¹)₂, or an alkyl, cycloalkyl, aryl, alkenyl,alkynyl or heterocyclic residue, each of which may be substituted orunsubstituted, and R¹⁴ is an alkylene residue; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1 wherein R¹ is Cl. 3.A compound having the following formula:

wherein R1 is a halogen atom, and R⁴ is H, a halogen atom, NO₂, CN, analkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclic,heteroalkenyl or heteroalkynyl residue, each of which may be substitutedor unsubstituted.
 4. The compound of claim 3 wherein R¹ is Cl and R⁴ isCH₃.
 5. A compound having the following formula:

wherein,

NH₂, R¹, R², and R³, which may be the same or different, each isindependently H, a halogen atom, NO₂, CN, OR¹³, SR¹³, N(R¹³)₂,

an alkyl, aryl, cycloaklyl, alkenyl, alkynyl, or heteroalkyl,heterocyclic, heteroalkenyl or heteroalkynyl residue, each of which maybe substituted or unsubstituted, R¹⁰ and R¹³ which may be the same ordifferent, each is independently H or an alkyl, cycloalkyl, alkenyl,alkynyl, aryl or heterocyclic residue, each of which may be substitutedor unsubstituted, and Z IS CH₃ or CHO; or pharmaceutically acceptablesalts thereof, with a proviso that when each of R² and R³ is hydrogen,and Z is CH₃, and X is NH₂, then R¹ is not NH₂, OCH₃ or CH₃.
 6. Thecompound of claim 5 wherein R¹ is Cl.
 7. A composition foresee treatingcancer comprising the compound of claim 1, 2, 3, 4, 5, or 6 and apharmaceutically acceptable carrier.
 8. The composition of claim 7wherein the cancer is selected from the group consisting of ovarian,colon, breast, cervical, esophageal, glioblastoma, neuroblastoma,stomach, kidney, skin, lung, pancreatic, seminoma, melanoma, bladder,thyroid, myeloid and lymphoid.
 9. A method of treating an animalsuffering from cancer comprising administering to the animal sufferingfrom cancer an effective amount of the compound of claim 1, 2, 3, 4, 5,or
 6. 10. The method of claim 9 wherein the cancer is selected from thegroup consisting of ovarian, colon, breast, cervical, esophageal,glioblastoma, neuroblastoma, stomach, kidney, skin, lung, pancreatic,seminoma, melanoma, bladder, thyroid, myeloid and lymphoid.
 11. Acompound having the following formula:

wherein R¹ is a halogen atom, and Z is either CHO or CH₃.
 12. Thecompound of claim 1 wherein R¹ is Cl.
 13. The composition of claim 8wherein Y is CO₂CH₃, CO₂H, CO₂(CH₂)₃CH₃, CONH₂, CO₂CH₂CH₂CH(CH₃)₂, orCO₂(CH₂)₇CH₃, R⁴ is CH₃ or H, R⁶ is H, and R¹⁰ is CH₃, or CH₃(CH₂)₂. 14.A method of treating HIV infection, comprising administering to ananimal suffering from such an infection an effective amount of acompound having the following formula:

wherein,

NH₂, Y is H, OR¹¹, SR¹¹, N(R¹¹)₂, NR¹¹N(R¹¹)₂, a halogen atom, NO₂, CN,

an alkyl, aryl, cycloalkyl, alkynyl, alkenyl or heterocyclic residue,each of which may be substituted or unsubstituted, R1, R2, R³, R⁴, R⁵,R⁶, R⁷, and R⁸, which may be the same or different, each isindependently H, a halogen atom, NO₂, CN, OR¹³, SR¹³, N(R¹³)₂,

an alkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclic,heteroalkenyl or heteroalkynyl residue, each of which may be substitutedor unsubstituted, R⁹ is H,

an alkyl, cycloalkyl, aryl, alkenyl, alkynyl or heterocyclic residue,each of which may be substituted or unsubstituted, R¹⁰, R¹¹ and R¹³,which may be the same or different, each is independently H or an alkyl,cycloalkyl, alkenyl, alkynyl, aryl or heterocyclic residue, each ofwhich may be substituted or unsubstituted, R¹² is H, N(R¹¹)₂, OR¹¹,SR¹¹, NR¹¹N(R¹¹)₂, OR¹⁴N(R¹¹)₂, or an alkyl, cycloalkyl, aryl, alkenyl,alkynyl or heterocyclic residue, each of which may be substituted orunsubstituted, and R¹⁴ is an alkylene residue; or a pharmaceuticallyacceptable salt thereof.
 15. The method of claim 14 wherein Y is CO₂CH₃,CO₂H, CO₂(CH₂)₃CH₃, CONH₂, CO₂CH₂CH₂CH(CH₃)₂, or CO₂(CH₂)₇CH₃, R⁴ is CH₃or H, R⁶ is H, and R¹⁰ is CH₃, or CH₃(CH₂)₂.
 16. The method of claim 14or 15 further comprising administering an effective amount of3′-azido-3′-deoxythymidine.
 17. A composition for treating an HIVinfection in an animal suffering from such an comprising: apharmaceutically acceptable carrier and an effective amount of acompound having the following formula:

wherein

Y is H, OR¹¹, SR¹¹, N(R¹¹)₂, NR¹¹N(R¹¹)₂, a halogen atom, NO₂, CN,

an alkyl, aryl, cycloalkyl, alkynyl, alkenyl or heterocyclic residue,each of which may be substituted or unsubstituted, R¹, R², R³, R⁴, R⁵,R⁶, R⁷, and R⁸, which may be the same or different, each isindependently H, a halogen atom, NO₂, CN, OR¹³, SR¹³, N(R¹³)₂,

an alkyl, aryl, cycloalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclic,heteroalkenyl or heteroalkynyl residue, each of which may be substitutedor unsubstituted, R⁹ is H,

an alkyl, cycloalkyl, aryl alkenyl, alkynyl or heterocyclic residue,each of which may be substituted or unsubstituted, R¹⁰, R¹¹ and R¹³,which may be the same or different, each is independently H or an alkyl,cycloalkyl, alkenyl, alkynyl, aryl or heterocyclic residue, each ofwhich may be substituted or unsubstituted, R¹² is H, N(R¹¹)₂, OR¹¹,SR¹¹, NR¹¹N(R¹¹)₂, OR¹⁴N(R¹¹)₂, or an alkyl, cycloalkyl, aryl, alkenyl,alkynyl or heterocyclic residue, each of which may be substituted orunsubstituted, and R¹⁴ is an alkylene residue; or a pharmaceuticallyacceptable salt thereof; with a proviso that the compound is notlavendamycin or streptonigrin.
 18. The composition of claim 17 wherein Yis CO₂CH₃, CO₂H, CO₂(CH₂)₃CH₃, CONH₂, CO₂CH₂CH₂CH(CH₃)₂, orCO₂(CH₂)₇CH₃, R⁴ is CH₃ or H, R⁶ is H, and R¹⁰ is CH₃, or CH₃(CH₂)₂. 19.The composition of claim 17 or 18 further comprising an effective amountof 3′-azido-3′-deoxythymidine.
 20. A method of treating an animal havinga tumor comprising administering to the animal an effective amount ofthe compound of claims 1, 2, 3 or
 4. 21. A method of preparing aquinoline dione having a structure

comprising reacting a 1-silyloxy-azadiene having the following formula:

with a bromoquinone having the following formula:

wherein

R¹, R², and R³, which may be the same or different, each isindependently H, a halogen atom, NO₂, CN, OR¹³, SR¹³, N(R¹³)₂,

an alkyl, aryl, cycloaklyl, alkenyl, alkynyl, or heteroalkyl,heterocyclic, heteroalkenyl or heteroalkynyl residue, each of which maybe substituted or unsubstituted.